Mouse anti myc tag

HeLa cells were cultured on coverslips in 24-well plates overnight. After transfection or/and infection, cells were harvested at the indicated times. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20min at room temperature. After being blocked with 3% bovine serum albumin (BSA) for 30min, cells were incubated with primary antibodies diluted in 1% BSA at 4°C overnight and secondary antibodies diluted in 1% BSA at room temperature for another 1h. Cells were mounted with Fluoroshield (Sigma) and examined by using a Leica confocal microscope after staining with 1 mg/ml 4’,6-diamidino-2-phenylindole (DAPI) in PBS. The primary antibodies used were as follows: goat anti-TIA-1 (1:200, Santa Cruz), goat anti-HPIV3 (1:1000, Abcam), rabbit anti-TIA-1 (1:500, ABclonal), rabbit anti-G3BP (1:500, ABclonal), rabbit anti-eIF4A (1:500, ABclonal), rabbit anti-eIF4E (1:500, ABclonal), rabbit anti-eIF4G (1:200, CST), rabbit anti-phosphorylated eIF2α (1:200, CST), mouse anti-G3BP (1:500, BD Bioscience), mouse anti-HA tag (1:2000, Sigma), mouse anti-Flag tag (1:1000, Sigma), and mouse anti-Myc tag (1:200, Santa Cruz). The secondary antibodies used were as follows: Alexa Fluor 647 donkey anti-goat IgG (1:1000, Invitrogen), Alexa Fluor 488 donkey anti-rabbit IgG (1:1000, Invitrogen), and Alexa Fluor 594 donkey anti-mouse IgG (1:1000, Invitrogen).

Protein was extracted with RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 10% glycerol) containing protease inhibitors and phosphatase inhibitors, and protein concentration was measured by Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Following separation by SDS-polyacrylamide gel electrophoresis, proteins were transferred onto PVDF membrane (Millipore, Billerica, MA, USA). Primary antibodies used in this study were as follows: rabbit anti-Sufu (C54G2; Cell Signaling Technologies, Danvers, MA, USA), mouse anti-α-SMA (ab7817; Abcam, Cambridge, UK), rabbit anti-FN (ab2413; Abcam, Cambridge, UK), mouse anti-myc tag (Santa Cruz Biotechnology, CA, USA), and rabbit anti-β-actin (13E5; Cell Signaling Technologies, Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rabbit (Sigma, USA) or anti-mouse IgG (GE, USA) was used as secondary antibody. Protein bands were detected using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, USA), and the densities of protein bands were measured using the Quantity One software.

Infected or transfected cells were harvested and lysed in cold TNE buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 2 mM EDTA [pH 8.0], 0.1% 2-mercaptoethanol and protease inhibitor cocktail). After incubation on ice for 30 min, cell lysates were centrifuged at 13,000 rpm for 30 min at 4°C. The clarified supernatant was mixed with 5XSDS-PAGE loading buffer, boiled at 100°C for 10 min and then subjected to 12% sodium dodecyl sulfate-polyacrylamide gel. The primary antibodies used were as follows: mouse anti-HPIV3 (1:2500, Abcam), rabbit anti-β-actin (1:1000, Proteintech), mouse anti-Myc tag (1:2500, Santa Cruz), mouse anti-HA tag (1:10000, Sigma), mouse anti-Flag tag (1:2500, Sigma), mouse anti-cofilin (1:2500, Proteintech), and rabbit anti-p-cofilin (1:1000, Cell Signaling Technology). HRP-conjugated goat anti-mouse IgG (1:5000, Sigma) and HRP-conjugated goat anti-rabbit IgG (1:5000, Sigma) were used as secondary antibodies.