Mouse anti cofilin

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.

Five days after lentiviral infection, cells were harvested and lysed in 2× protein lysis buffer (10 mmol/L EDTA, 100 mmol/L Tris‐Hcl (pH 6.8), 4% SDS and 10% Glycine). The suspension was collected after centrifugation at 12,000g for 15 min at 4°C.Equal amounts (30 μg) of proteins were run on a 10% SDS‐PAGE and subjected to electrophoresis at 50 V for 3 h on a 10 % separating‐gel. The gel was transferred to polyvinylidene difluoride (PVDF) membrane, which was then blocked with TBST containing 1% bovine serum albumin (BSA) in for 1 h and incubated with primary antibodies overnight and then incubated with secondary antibodies for 2 h. Primary antibodies used were as follows: rabbit anti‐PAK4 (1:1000, Proteintech Group, Inc., 14685‐1‐AP), rabbit anti‐LIMK1 (1:1000, Proteintech Group, Inc., 19699‐1‐AP), rabbit anti‐p‐LIMK1 (1:1000, SAB, 11126), rabbit anti‐p‐cofilin (1:1000, SAB, 11139), mouse anti‐Cofilin (1:1000, Proteintech Group, Inc., 66057‐1‐Ig) and rabbit anti‐GAPDH (1:1000, Proteintech Group Inc., 10494‐1‐AP).