Mouse anti hpiv3

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.

Infected cells were washed, scraped into cold phosphate-buffered saline (PBS), pelleted by centrifugation at 13,000 rpm for 1 min, and then 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) for 30 min. Cell lysis was achieved through 30 rounds of Dounce homogenization on ice, and cell lysates were centrifuged at 13,000 rpm for 30 min at 4°C. The clarified supernatant was mixed with 5x SDS-PAGE loading buffer, boiled at 100°C for 10 min, and then subjected to 12% sodium dodecyl sulfate-polyacrylamide gel. The samples were then electroblotted onto a nitrocellulose membrane, blocked with skim milk in phosphate-buffered saline with Tween 20 (1/1,000 Tween 20) for 30 min at room temperature, and subsequently incubated with primary antibodies for 1 h and secondary antibodies for 45 min. The primary antibodies used were mouse anti-HPIV3 (1 : 2500, Abcam), rabbit anti-PI4KB (1 : 2500, Sigma), and rabbit anti-β-actin (1 : 1000, Proteintech). HRP-conjugated goat anti-mouse IgG (1 : 5000, Sigma) and HRP-conjugated goat anti-rabbit IgG (1 : 5000, Sigma) were employed as secondary antibodies.