Agarose

Total protein extraction and measurement of concentration were performed as described under Western blotting method. One thousand micrograms of total protein were incubated with 10 μl of Anti-ITGA6 antibody conjugated with agarose (Santa Cruz Biotechnology, Santa Cruz, CA, Cat.# sc-374057) overnight at 4°C. The pellet was collected by centrifugation at 3,000rpm for 30s and washed 3 times with cold PBS. Then the pellet was resuspended in electrophoresis sample buffer and applied to SDS-PAGE electrophoresis as described under Western blotting method.

At the indicated time points of MLR culture cells, T cells were isolated and were harvested and protein lysates were prepared by washing cells in PBS and lysing them in lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM MgCl₂, 10% glycerol, and 1% NP-40 supplemented with 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and Protease Inhibitor Cocktail (Thermo Fisher Scientific), according to previously described methods [20 (link)]. Samples were analyzed by SDS-PAGE transferred to nitrocellulose membranes, followed by immunoblot with the indicated antibodies. For in vitro kinase reactions, immunoprecipitations were performed with mAb against Cdk2 that was conjugated to agarose (Santa Cruz) followed by in vitro kinase reactions with histone H1 as the exogenous substrate, as previously described [20 (link)]. Reactions were analyzed by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane, and exposed to film.

After stimulation of the cells with VEGF-A (50 ng/mL) for indicated time points, cells were lysed with IP-lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 0.25% NP40, 2.5% glycerol, protease inhibitor cocktail, 1 mM β-glycerophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate). Total protein lysates were precleared with normal IgG conjugated with agarose (Santa Cruz) for 1 hr at 4°C, then proceeded for co-IP with specific antibodies (Rab5A, Rab 11 and Rab7) and Protein A/G bead (Thermo Fisher) for overnight at 4°C. After washing with IP-lysis buffer with reduced detergent (0.025% NP40), proteins co-immunoprecipitated with Rab family antibodies were analyzed by western blotting.