Antibody News

Measuring Autophagic Flux with LC3 protein levels: The do's and don'ts

Tuesday, August 21, 2018 - 08:50
Expression of p62/SQSTM1 in HeLa cells mock- or chloroquine-treated, ICC

By Christina Towers, PhD.

Autophagy is a dynamic cellular recycling process that can be influenced by many different external and internal stimuli. The most commonly used assay to measure autophagy is a western blot for the autophagic membrane-specific protein, LC3. While this assay is relatively simple, if not controlled properly, the results can easily be misinterpreted. Indeed, the most accurate way to measure autophagy is with an autophagic flux assay defined as the new formation of double-membrane vesicle-like structures called autophagosomes and their...

Knockout Validation and the Reproducibility Crisis

Tuesday, August 14, 2018 - 09:34
Vimentin expression in K562 leukemia cell line ICC

By Colin O'Neill, 2018 Novus Intern

The specificity and affinity of an antibody for its target protein—and only its target is valued as an indicator of its experimental reliability. The frustrating ability of an antibody to bind to multiple proteins has prompted a reproducibility crisis in the life science community. Specifically, if an antibody varies in specificity between batches, attempts to reproduce significant findings with that antibody may fail. With samples, time, and funding at stake, scientists have developed novel validation techniques to ensure antibodies solely bind to their intended targets to avoid false positives and ambiguous results. Nature's 2015 article...

Eat responsibly: Epigenetic downregulation of Ankrd26 gene by long-term high-fat intake promotes obesity and inflammation

Tuesday, August 7, 2018 - 08:38
DNTM3A expression in human cell line A-431 ICC

By Jamshed Arslan Pharm.D.

White adipose tissue (WAT) represents the primary site to store energy in humans. WAT’s endocrine regulation of energy balance is controlled by nutritional status, exercise, and hormones like insulin. Partial inactivation of a gene highly expressed in WAT, called Ankrd26, induces obesity and diabetes in mice. Moreover, feeding mice high-fat diet (HFD) has been suggested to cause Ankrd26 promoter hyper-methylation, an epigenetic mechanism of gene silencing. Working along these lines, a team of researchers from Italy and USA set out to elaborate how HFD can epigenetically...

You complete me: Natural killer cells need TGF-beta inhibition to effectively combat cancers

Tuesday, July 31, 2018 - 08:22
TGF-beta signaling pathway

By Jamshed Arslan Pharm.D.

Natural killer (NK) cells are lymphocytes of the innate immune system that were first discovered for their “natural” ability to kill cancer cells. To use NK cells as anti-cancer therapy, they are co-cultured with feeder cells that are sensitive to attack by NK cells. The killing of feeder cells in turn generates highly efficient tumor-killing NK cells. However, a major challenge in using these ex vivo expanded NK cells as anti-cancer therapy is the highly immunosuppressive tumor microenvironment, partly because the cancer cells, myeloid derived suppressor cells and other stromal cells produce excessive amounts of...

Meningeal lymphatics: recent discovery defying the concept of central nervous system 'immune privilege'

Tuesday, July 24, 2018 - 08:57
Regulation Of Lymphangiogenesis Pathway Bioinformatics

By Jennifer Sokolowski, MD, PhD.


Identification and characterization of meningeal lymphatics

The recent discovery of a lymphatic system in the meninges surrounding the brain and spinal cord has spurred a surge of interest in and has redefined our understanding of immunity in the central nervous system (CNS).1,2 The lymphatic system in the brain is composed of the glymphatic system and meningeal lymphatic vessels.3 The glymphatic system involves convective flow of cerebral spinal fluid and interstitial fluid and uses para-arterial flux to clear solutes and metabolites from the brain parenchyma. In concert, the meninges harbor bona...

Monitoring Autophagy in Neurons

Tuesday, July 17, 2018 - 08:49
Beclin 1 expression in human cerebellum, Purkinje cells IHC

By Christina Towers, PhD.

Autophagy is a critical cellular process used by most cells in the body to recycle nutrients and prevent harmful buildup of damaged proteins. It is particularly important in the brain, where a handful of pathologies have been linked to autophagy dysregulation. Conditional neuronal knock out of the core autophagy gene, ATG7, results in viable mice that eventually succumb to neurodegeneration accompanied by an accumulation of ubiquitin protein aggregates1, 2. Likewise, decreased levels of functional autophagy have been linked to the three most common neurodegenerative diseases including...

Antigen-loss relapse after successful CAR-T therapy; What do we do now?

Tuesday, July 10, 2018 - 08:30
Acute lymphoblastic leukemia disease bioinformatics

By Jacqueline Carrico, BS, MD

Tumor cell mechanisms driving tolerance to CAR-T

Despite very promising results of CAR-T therapy in acute lymphoblastic leukemia, B-ALL, antigen-loss relapse has arisen as a major challenge for maintaining long term remission. Here we will review the potential mechanisms for antigen-loss relapse and some novel strategies to combat this problem.

One possible mechanism for antigen loss is downregulation of expression of the target protein in the malignant cells. Constant therapeutic stress from the highly-targeted CAR T-cells may lead to this phenomenon. Additionally, tumor...

Developmental regulator Daam2 promotes glial cell tumors by degrading Von Hippel-Lindau protein

Tuesday, July 3, 2018 - 08:35
GFAP expression neuron glia mixed culture ICC

By Jamshed Arslan Pharm.D.

Glioblastoma is an aggressive type of cancer that forms from the star-shaped glial cells of the central nervous system, called astrocytes. Intriguingly, several genes linked to glioblastomas, including the gene for the tumor suppressor Von Hippel-Landau protein (VHL), do not exhibit mutations. VHL prevents tumorigenesis by binding and modulating the function of hypoxia-inducible factor HIF-1 alpha and hydroxylated serine-threonine kinase Akt. Developmental processes that maintain cells in a...

MAPK Signaling Links Autophagy and Inflammation

Tuesday, June 26, 2018 - 09:53
Autophagy signaling pathway

By Christina Towers, PhD.

MAPK Signaling and Disease

Mitogen-activated protein kinases (

Autophagy Inhibition in Cancer: Clinical Trials Update

Tuesday, June 19, 2018 - 10:43
Autophagy signaling pathway

By Christina Towers, PhD.

Autophagy mediates the recycling of damaged cellular material into building blocks like amino acids and other necessary nutrients that can fuel metabolism and cell growth, especially under nutrient depleted conditions. There are currently over 60 clinical trials reported on ClinicalTrials.gov that are either completed or on-going using autophagy inhibition, mostly in combination with other targeted therapies. Autophagy has a pro-tumorigenic role in established tumors and consequently the vast majority of clinical trials focus on autophagy inhibition in cancer. Over the last decade, a dozen Phase I and I/II clinical trials...

Adenosine Inhibits T cell Tumor Infiltration: KCa3.1, a New Anticancer Target

Tuesday, June 12, 2018 - 11:47
CD8 alpha antibody, ICC

By Yoskaly Lazo-Fernandez, PhD

Role of Adenosine in the Tumor Microenvironment a Target for Cancer Therapy

The tumor microenvironment (TME) tends to be concentrated in the purine nucleoside adenosine, a direct result of the hypoxia normally associated with cancer. Extracellular adenosine binds to its receptor, A2A receptor (A2AR), in the surface of lymphocytes and other immune cells resulting in anti-inflammatory and immunosuppressive responses1 which correlate with higher tumor progression and poor prognosis in cancer patients2. Extracellular adenosine accumulation...

Crosstalk Between Oxidative Stress and Autophagy

Tuesday, June 5, 2018 - 11:10
Autophagosome and lysosome fusion

By Christina Towers, PhD.

Role of Reactive Species in Cellular Function

Oxidative stress is a byproduct of an imbalance between oxidants and antioxidants present in the cell resulting in dysfunctional redox signaling. This disproportion is caused by naturally occurring reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can be derived from either extracellular sources or intracellularly as byproducts of essential cellular processes like metabolism. These species oxidize and remove electrons from the molecules they interact with including many kinds of biomolecules which can be detrimental to overall cellular function1. For example, ROS can induce...

Novel Approaches to Improve Efficacy and Safety of CAR-T Therapy

Tuesday, May 29, 2018 - 08:44
CAR-T Therapy

By Jacqueline Carrico, BS, MD Candidate

Given the rapid advances in CAR-T therapy, there have been major efforts to improve the specificity and safety of these therapies. Very few targets exist that are only expressed in the malignant cell population, resulting in on-target/off-tumor toxicities. Most have been minor and controllable; however, several trials have been terminated due to severe and life-threatening toxicities. There is increasing concern about this problem with dually targeted therapies, such as dual-CAR or tandem CAR.

Synthetic Notch Receptors

One approach to increasing on-tumor specificity is the development of CAR-T cells that express synthetic...

Lysosomal Dysfunction is Linked to Exosomal Secretion

Tuesday, May 22, 2018 - 09:50
CAR-T Poster

By Christina Towers, PhD.

Lysosomal Dysfunction and Disease

Lysosomes are highly acidic organelles that are critical for cellular function and indispensable for degradative pathways like autophagy and endocytosis.  There are a number of different diseases that have been associated with lysosomal dysfunction, the most detrimental being neurological disorders including Huntington's Disease, Alzheimer's disease, and Parkinson's disease.  Other rare neurological diseases like Niemen-Pick disease Type C (NPC) have been directly linked to familial-mutations in endolysosomal genes.  All of these disorders are attributed to a buildup of the detrimental...

Targeting Success in CAR-T Therapy for Solid Tumors

Tuesday, May 15, 2018 - 09:40
CAR-T poster

By Jacqueline Carrico, BS, MD Candidate

Targeting Success in CAR-T Therapy for Solid Tumors

Developing successful CAR-T therapy requires identification of specific tumor-associated antigens, as the primary target for CAR-T binding and activation. Some solid tumors have well-characterized oncogenes which play a pivotal role in tumor cell proliferation, migration, and survival. These oncogenes are ideal targets for CAR-T therapy, particularly when the oncogene is expressed at low levels in normal tissues.

Epidermal Growth Factor Receptor (EGFR)

The tyrosine kinase receptor EGFR, is aberrantly expressed in non-small cell lung cancer (NSCLC),...

Mitochondrial ATPase inhibitory factor 1 (IF1) provides an explanation of cancer growth in anoxia or pseudo-anoxia

Tuesday, May 8, 2018 - 09:29
ATPase inhibitor factor 1 antibody ICC

By Jamshed Arslan Pharm.D.

Adenosine triphosphate (ATP) is the major life’s energy-carrying molecule. It is mainly produced by mitochondrial ATP synthase (Complex V) through oxidative phosphorylation (Oxphos). For example, Oxphos-dependent oxidation of a glucose molecule generates about 30 molecules of ATP. In Oxphos, respiratory chain (r.c.) complexes catalyze the transfer of electrons from energy-rich molecules (NADH or FADH2) to oxygen (O2) and...

TGF-beta for treating degenerative intervertebral disc disease

Tuesday, May 1, 2018 - 09:32
TGF-beta poster

By Jamshed Arslan Pharm.D.

Our upright posture and balance depend on a jelly-like material, called nucleus pulposus (NP), in the middle of intervertebral discs. NP cells protect us from disc degeneration by maintaining optimal amounts of proteoglycans (proteins bonded to glycosaminoglycans) in the NP matrix. This process can be facilitated by TGF-beta, which stimulates the synthesis of sulfated glycosaminoglycan (sGAG) and chondroitin sulfate proteoglycan 1 in the NP cells. The synthesis of sGAG depends on chondroitin polymerizing factor (ChPF), an enzyme that extends the chondroitin sulfate (CS) backbone in sGAG. However, the...

Autophagy independent roles of the core ATG proteins

Tuesday, April 24, 2018 - 11:24
ATG5 ICC Antibody

By Christina Towers, PhD.

Autophagy and ATG Proteins

Autophagy is a nutrient recycling process that cells use to fuel metabolism, particularly in response to nutrient deprivation.  It is critical for removal of damaged proteins as well as clearing certain infections.  It is a complex process that involves over 20 core autophagy genes (ATGs), most of which were originally identified in yeast but the mammalian homologs have now been well characterized.

The formation of a double membrane vesicle termed the phagophore is initiated by the ULK complex where the protein kinases ULK1 and...

Immunity’s flipside: Microglia promote Alzheimer’s pathology during inflammation

Tuesday, April 17, 2018 - 11:04
ICC Amyloid beta antibody

By Jamshed Arslan Pharm.D.

Microglia are brain's macrophages. In Alzheimer's disease (AD), microglia clear up protein aggregates called amyloid beta plaques. The connection between immune activation and AD is unclear, but a major sensor for danger-signals, called NLRP3 inflammasome, is known to be activated in the brains of patients and transgenic mice (APP/PS1) that overproduce amyloid beta.1 Activated NLRP3 inflammasome leads to the release of pro-inflammatory cytokine (IL-1 beta...

Nuclear LC3: Why is it there and what is it doing?

Tuesday, April 10, 2018 - 09:17
hHpr1-p84-Thoc1 Antibody

By Christina Towers, PhD.

Cells use the complex process of autophagy to degrade and recycle cytoplasmic material.  There are over 20 proteins that have been implicated in this process and appropriately named core autophagy genes (ATGs).  Most of these were originally discovered in yeast, arguably the most famous being ATG8.  In higher eukaryotes ATG8 evolved into a family of proteins known as the GABA type A receptor-associated protein (GABARAP), which includes the Microtubule Associated Protein 1 light chain 3 (MAP1LC3), better known...

CAR-T Cell Therapy: Refining the Approach in Solid Tumors

Tuesday, April 3, 2018 - 08:30
CAR-T Cell Killing

By Jacqueline Carrico, BS, MD Candidate

Chimeric antigen receptor T-cells, better known as CAR-T cells, are being used as a novel anticancer therapy. CAR-T cells are engineered T-cells which express a modified antigen-receptor. Each chimeric antigen receptor contains 3 domains: an extracellular binding domain, a transmembrane hinge domain, and an intracellular activation or costimulatory domain. The extracellular portion is the single chain variable fragment (scFv), made up of an antibody-derived heavy chain and light chain, which ultimately recognize specific tumor antigens. The intracellular activation domain allows T-cell activation upon...

Killing two birds with one stone: Treating inflammation and cancer by inhibiting prolyl-4-hydroxylase-1

Tuesday, March 27, 2018 - 10:41
Bi-directional Cross-talk Between HIF1-alpha and NF kappa B

By Jamshed Arslan Pharm.D.

The cell’s oxygen-sensing machinery comprises prolyl-4-hydroxylases (P4Hs 1-3, PHDs 1-3, or EGLN 1-3) and their canonical target hypoxia-inducible factors (HIFs). When oxygen levels are low, PHDs become functionally inactive, leading to HIFs’ stability. PHD1 is thought to provide a link between hypoxia and inflammation, partly because it influences the prototypical proinflammatory transcription factor NF-kB in such a way that suppressing PHD1 reduces inflammation....

Getting Physical: Link between Lipid Metabolism and Hypoxia Target Genes

Tuesday, March 20, 2018 - 11:16
Autophagy Mechanisms

By Jamshed Arslan Pharm.D.

von Hippel-Lindau (VHL) disease is associated with tumors arising in multiple organs. Activation of hypoxia-inducible factor (HIF)-alpha underlies the VHL disease pathogenesis. In normoxia, VHL tumor-suppressor protein (pVHL) and E3 ubiquitin ligase lead to proteosomal degradation of HIF-alpha. In hypoxia, HIF-alpha escapes degradation, partly because pVHL binding to HIF-alpha depends on a posttranslational modification (hydroxylation of proline residues) on HIF-alpha that only occurs in normoxia. The exact role of pVHL in tumor hypoxia, when HIF-alpha is stabilized, is poorly...

Autophagy inhibition in pediatrics: One physician-scientist’s brave decision

Tuesday, March 13, 2018 - 13:05
Autophagy Pathway

By Christina Towers, PhD.

The current time from when a discovery is first made on the bench to when that discovery might translate into an approved therapy in cancer patients is an astounding 10-15 years. Scientists and clinicians alike face a daunting uphill battle to find novel targeted therapies that can improve a patient’s outcome yet still maintain minimal adverse side effects.  However, recently Dr. Jean Mulcahy Levy, a pediatric neuro-oncologist and physician-scientist, found a way to bypass some of these hurdles.  In 2012 she was working as a research-fellow at the University of Colorado in the laboratory of Dr. Andrew Thorburn, an expert in the field of...

Cleaner gone bad: Autophagy regulates motor neuron loss in spinal muscular atrophy

Wednesday, March 7, 2018 - 11:10
Autophagy Mechanisms

By Jamshed Arslan Pharm.D.

Neuromuscular disorders affect the peripheral nervous system and muscles. Spinal muscular atrophy (SMA) is one such incurable disease in which muscles fail to receive signals from the spinal motor neurons (MNs), and consequently, weaken due to inactivity. MN degeneration and muscle atrophy lead to the premature death of the victims. Like most of the neuromuscular disorders, SMA is genetic, and its genetic causes are known: the inactivation of survival motor neuron 1 (SMN1) gene, which reduces the...

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