Anti gja1

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).

Embryos were fixed with MEMFA or Dent’s fixative solution overnight at 4°C, and RPE1 cells were fixed with 4% PFA solution for 20 min at room temperature. Fixed embryos or RPE1 cells were incubated in a blocking solution (10% FBS and 2% DMSO in 1×TBS with 0.1% Triton X-100) for 30 min at room temperature to block non-specific binding. Immunostaining was performed with the following antibodies at 1:50–500 dilutions in blocking solution for 1–2 hr at room temperature: anti-DDDD-K (Abcam, Cambridge, UK, ab1162), anti-GFP (ThermoFisher Scientific, A10262), anti-HA (Santa Cruz, sc-7392), anti-RFP (Abcam, ab62341), anti-acetylated tubulin (Sigma Aldrich, T7451), anti-γ-tubulin (Abcam, ab191114), anti-MyHC (DSHB, IA, USA, A4.1025), anti-CP110 (Proteintech, IL, USA, 12780–1-AP), anti-BBS4 (Proteintech, 12766–1-AP), anti-TGN46 (Abcam, ab50595), anti-Arl13b (Proteintech, 17711–1-AP), anti-TTBK2 (Proteintech, 15072–1-AP), anti-IFT20 (Proteintech, 13615–1-AP), anti-IFT88 (Proteintech, 13967–1-AP), anti-Rab8a (Cell Signaling, MA, USA, #6975), anti-Rab11 (Cell Signaling, #5589), and anti-GJA1 (both ThermoFisher Scientific, PA1-25098, 13–8300). Fluorescence labeling was performed using DAPI (1:10,000, ThermoFisher Scientific, D1306), and Alexa Fluor 488-, 555-, and 633-conjugated secondary antibodies (1:300, all ThermoFisher Scientific).