Acrylamide bis 37.5 1

We lysed cells in RIPA buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF] for 60 min on ice, and collected supernatants after centrifugation at maximum speed for 30 min. We determined protein concentrations by Bradford Assay (Bio-Rad). Before gel loading, we boiled 40 µg of protein per sample for 10 min with sodium dodecyl sulfate (SDS)-loading buffer. We separated cell lysates by SDS-PAGE on an 8% gel (Acrylamide/Bis 37.5:1; Bio-Rad) in 1× SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS), and transferred proteins to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 100 V for 1.5 h using a wet-tank electrotransfer system (Bio-Rad). We blocked membranes in 5% milk in 1× TBST (144 mM NaCl, 20 mM Tris, 0.05% Tween 20, pH 7.6) for 1 h and washed them with 1× TBST thrice, each for 5 min. We incubated membranes with the primary antibody anti-rabbit PRPF40B (Abcam) or anti-mouse GAPDH (Sigma) in 1× TBST at 4°C overnight followed by three washes with 1× TBST. We incubated membranes with secondary anti-mouse/rabbit IgG-HRP antibodies (BioLegend, Sigma, Santa Cruz Biotechnology) diluted in 1× TBST with or without 5% milk. After three washes, we detected proteins by Western Lightning Plus ECL (PerkinElmer).

Radiolabeled RNA was incubated in JSL1 nuclear extract under similar conditions described for the EMSAs. Reactions were incubated for 20 min at 30°C, crosslinked using UV light (254 nm) for 20 min on ice, and digested with 2 ug (final concentration) of RNase T1 and RNase A each for 20 min at 37°C. Reactions were analyzed under denaturing conditions on a 12% gel (Acrylamide/Bis 37.5:1, BioRad), and visualized by autoradiography. Immunoprecipitation following crosslinking was done as described in ref 18 using the following antibodies: CELF2 (UFl Hybridoma Lab, HL1889); HuR (Santa Cruz, sc-5261); hnRNP C (Abcam, ab10294); and hnRNP H (Abcam, ab10374).