Phoshostop phosphatase inhibitor tabs

Western blot lysates were produced in RIPA buffer (Pierce #89900) supplemented with cOmplete Mini EDTA-free protease inhibitor cocktail and PhoshoStop phosphatase inhibitor tabs (Roche) and 1 mM DTT at 4 °C. Lysates were cleared by centrifugation at 16,000 x g for 25 min at 4 °C. Proteins were separated using 3–8% or 7% Tris-Acetate, or 4–12% Bis-Tris precast mini gels (Life Technologies) and transferred to PVDF membranes at 70 V for 2 h or overnight (Immobilon P, Millipore). Ponceau S staining was used to verify even transfer (Sigma Aldrich). Membranes were optionally cut to detect multiple proteins. Blocking and antibody incubations were then performed with either 5% non-fat milk or bovine serum albumin (fraction V) in PBST or TBST. Bands were visualized with HRP-conjugated secondary antibodies (DAKO) followed by the application of a chemiluminescent reagent (Thermo Scientific). Stripping with Restore^TM PLUS stripping buffer (Thermo Scientific) and reprobing was performed in some cases when the subsequent primary was of a different species; however, blots were never stripped more than once. Densitometry was performed in ImageJ on unaltered images.

Cells were lysed in co-immunoprecipitation buffer (20 mM HEPES-KOH pH 7.9, 200 mM NaCl, 10% (v/v) glycerol, 0.1% Triton X-100, 1 mM DTT) supplemented with cOmplete Mini EDTA-free protease inhibitor cocktail and PhoshoStop phosphatase inhibitor tabs (Roche) at 4 °C. Lysates were cleared by centrifugation at 16,000 x g for 25 min at 4 °C. Simultaneously, 4 or 5 ug of appropriate antibodies or IgG control proteins were bound to protein G Dynabeads^TM as per manufacturer instructions (Life Technologies). Equal portions of cleared lysates were added to antibody or IgG bound Dynabeads^TM and incubated for 3 h or overnight at 4 °C. Beads were washed with co-immunoprecipitation buffer at 4 °C 4x for co-immunoprecipitation and 6x for immunoprecipitation, followed by elution with a 2:1 mixture of 50 mM Glycine, pH 2.8 and 1x NuPAGE^TM LDS sample buffer (Life Technologies) at 70 °C for 10 min.