Mouse anti ha antibody ha 11 clone 16b12

The following commercially available antibodies were used: mouse anti-HA antibody HA.11 (clone 16B12, Biolegend, Cat# 901503, RRID:AB_2565005), rabbit anti-DYKDDDDK (FLAG) tag antibody (clone D6W5B, Cell Signaling Technology Cat# 14793, RRID:AB_2572291), HRP-coupled rabbit anti-E tag antibody (Bethyl Laboratories Cat# A190-133P, RRID:AB_345222), HRP-coupled MonoRab rabbit anti-camelid VHH antibody (clone 96A3F5, GenScript Cat# A01860-200, RRID:AB_2734123), HRP-coupled mouse anti-HA-Tag (clone 6E2, Cell Signaling Technology Cat# 2999, RRID:AB_1264166), goat anti-mouse IgG Alexa Fluor Plus 647 (Thermo Fisher Scientific Cat# A32728, RRID:AB_2633277), goat anti-rabbit IgG Alexa Fluor Plus 647 (Thermo Fisher Scientific Cat# A32733, RRID:AB_2633282).

Proteins from cell lysates were resolved through 11% SDS-PAGE and analyzed by western blotting. In brief, cells were harvested 48 h after transfection and centrifuged at 300× g. Cell were then resuspended in a RIPA lysis buffer and incubated on a rolling wheel for 30 min at 4 °C. Clarified supernatants were then mixed with a 2X Laemmli buffer. Samples were boiled at 95 °C for 5 min before being loaded onto 11% acrylamide, and then transferred on an Immobilon FL-PVDF membrane (Millipore, Burlington, MA, USA). The membranes were blocked in Odyssey Blocking Buffer (Li-COR, Lincoln, NE, USA) diluted 1:1 in TBS for 30 min at room temperature. Membrane probing was performed with a mouse anti-HA antibody (HA.11, clone 16B12; Biolegend, San Diego, CA, USA), a mouse or rabbit β-actin antibody (Li-COR); secondary antibodies used were goat anti-rabbit IRDye 800 and goat anti-mouse IRDye 680 (Li-COR). Proteins bands were visualized using the Li-COR Odyssey infrared imaging System (Li-COR).