Anti pbcbp1

Samples of interest were lysed in RIPA buffer (Cell Signaling 9806) containing protease inhibitor cocktail (Roche 11836170001), 1 mM PMSF, and phosphatase inhibitor (Pierce A32957), unless noted otherwise. Protein lysate concentrations were normalized using Pierce BCA (Thermo 23225), and then reduced and denatured in sample buffer (Thermo 39000). Lysates were separated on Bio-Rad 4%–20% Mini-PROTEAN TGX precast gels, transferred via Bio-Rad Trans-Blot Turbo onto PVDF membranes (Bio-Rad 1704272), blocked in TBS-T (TBS with 0.1% Tween-20) containing 5% BSA, and probed overnight at 4°C with primary antibody. The next day, blots were washed in TBS-T and probed with HRP-linked secondary antibody (Cell Signaling 7076; 7074; Streptavidin-HRP 3999S) for 1 hour. Signal detection was performed using ECL substrate (Thermo 34577; 34095) and a Bio-Rad ChemiDoc MP imaging system. Blot images were digitally processed and analyzed in ImageJ. The primary antibodies used were as follows: anti-actinin (isoforms 1–4) (Cell Signaling 6487), anti-HA (Cell Signaling 14793), anti-IGF2BP2 (MBL RN008P), anti-PBCBP1 (Abcam 74793), anti-PCBP2 (MBL RN250P), anti-SERBP1 (Abcam 55993), anti-TCAP (BD 612328), anti-FLAG (Cell Signaling 2368), anti-GAPDH (Cell Signaling 2118) and anti-MYOM (mMaC myomesin B4 was deposited to the DSHB by Perriard, J.-C.).