Ab51081

Cell extracts were resolved on a 10% SDS–PAGE gel and transferred to polyvinylidene fluoride membrane (Millipore, San Diego, CA) using a semidry apparatus. The membranes were blotted with the antibodies indicated in each figure, and bands were visualized using the ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, MA). Membranes were incubated with the following primary antibodies: anti-Rck/p54 (ab54611; Abcam, Cambridge, United Kingdom), anti-Ago2 (ab57113; Abcam), anti-Dcp1a (ab57654; Abcam), anti–β-actin (A2228; Sigma), anti-Matr3C (ab84422; Abcam), anti-Matr3N (ab51081; Abcam), PSF (sc-101137; Santa Cruz, Santa Cruz, CA), DDx5 (ab21696; Abcam), Importin-β (ab2811; Abcam) mouse anti–α-tubulin (T5168; Sigma-Aldrich), rabbit-LaminB1 (sc-20682; Santa Cruz). Secondary antibodies conjugated to horseradish peroxidase (GE Healthcare) were used at 1:10,000 dilutions and developed using ECL reagent and X-ray film. Densitometry measurements were performed using ImageJ.

Cells were fixed for 5 min in 3.7% paraformaldehyde (PFA) (Sigma-Aldrich) followed by 2 min permeabilization with 0.5% NP-40 (EMD Millipore). The coverslips were incubated with blocking solution (1% bovine serum albumin) for 1 h at RT. Primary antibodies used for immune staining were diluted in blocking solution and applied to the coverslips for 1 h at RT; this was followed by three short rinses in PBS. Primary antibodies were as follows: anti-Matr3C (ab84422; Abcam), anti-Matr3N (ab51081; Abcam), and mouse anti-S6K used to detected Hedls (sc-8418; Santa Cruz) at 1:200; rabbit anti-Rck/p54 (A300-461A; Bethyl Laboratories, Montgomery, TX) and mouse anti-PABP-1 (10E10, sc-32318; Santa Cruz) at 1:200. Next the coverslips were incubated with secondary antibodies, diluted 1:1000 in blocking buffer, and conjugated to Alexa Fluor 405, 488, or 546 (Invitrogen, Carlsbad, CA) for 1 h at RT. The coverslips were next stained with 4′,6-diamidino-2-phenylindole and rinsed with PBS before being mounted onto slides using Mowiol mounting media. Images were acquired at RT using a using an Olympus IX81 wide-field microscope with a 40× air objective lens attached to a Hamamatsu Orca-R2 cooled CCD camera. Pearson’s colocalization coefficients were determined using the Coloc2 plug-in in imageJ.