Anti α actin

Xenopus or human RPE1 immunoblotting samples were prepared with modified lysis buffer (TBS, 1% Triton X-100, and 10% glycerol with protease inhibitor). After removing fat and cellular debris, a sodium dodecyl sulfate (SDS) sample buffer with dithiothreitol (DTT) was added. Samples were loaded on SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Merck Millipore, MA, USA). Membranes were incubated in blocking solution (TBS, 0.05% Tween-20 with non-fat powdered milk) for 30 min at room temperature to block non-specific binding. Immunoblotting was performed with the following antibodies at 1:2,500–3,000 dilutions for either 1 hr at room temperature or overnight at 4°C: anti-DDDD-K (Abcam, ab1162), anti-α-actin (ThermoFisher Scientific, MA1-744), anti-α-tubulin (Abcam, ab15246), anti-GJA1 (ThermoFisher Scientific, PA1-25098), anti-HA (Santa Cruz, sc-7392), anti-Rab8a (Cell Signaling, #6975), and anti-Rab11 (Cell Signaling, #5589). Secondary labeling was performed using horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (1:3000, both ThermoFisher Scientific, 31430, 31460) for 1 hr at room temperature. Chemiluminescence was performed with SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific, 34076), and the membranes were imaged with an iBright imaging system (FL1000, ThermoFisher Scientific).

Human chondrocyte samples were prepared using a lysis buffer (TBS, 10% glycerol, 1% Triton X-100, protease inhibitor, and phosphatase inhibitor (Invitrogen)). After removing cell debris, SDS sample buffer containing dithiothreitol was added. Samples were resolved using SDS-PAGE, and then transferred to PVDF membranes (Merck Millipore). Transferred membranes were incubated in blocking solution (TBS, 0.05% Tween-20 with 5% nonfat powdered milk) for 30 min at room temperature to block nonspecific binding. Immunoblotting was performed using anti-α-actin (Thermo Fisher Scientific, Waltham, MA, USA) and anti-ITGBL1 (AbClon, Seoul, Republic of Korea, custom order) antibodies at 1:1000 ~ 1:3000 dilutions, overnight at 4 °C. The blots were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (both from Thermo Fisher Scientific) at 1:3000 for 1 h at room temperature. Chemiluminescence was performed using SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific) and imaged with an iBright imaging system (Thermo Fisher Scientific).