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![<b>Biological Strategies Validation. </b>Rh159 interferes with intracellular transport of NKG2DL.A) Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. B) Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. C) Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. D-E) Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH (D),](http://images.novusbio.com/fullsize/af1298_human-ulbp-2-5-6-affinity-purified-polyclonal-ab-8220249503219.jpg)
![<b>Biological Strategies Validation. </b>Rh159 interferes with intracellular transport of NKG2DL.A) Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. B) Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. C) Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. D-E) Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH (D),](http://images.novusbio.com/fullsize/af1298_human-ulbp-2-5-6-affinity-purified-polyclonal-ab-4420241542505.jpg)