Anti ift20

For determination of IFT20 expression in RCC4+pVHL-, RCC4-, RCC4 siCtl-, RCC4 siVDAC1-, RCC4 siLGMN- cells, cellular suspensions (1 × 10⁶ cells) were resuspended in 4% PFA for 15 min and washed 3 times in cold PBS/BSA 0.1%. Cell suspensions were then incubated with a polyclonal rabbit anti-IFT20 (1 µg/1 × 10⁶ cells; Proteintech) for 1 h at 4°C in PBS/BSA 0.1% buffer. After washing, cells were incubated with Alexa 488-conjugated secondary goat anti-rabbit antibody (µg/1 × 10⁶ cells;) for 30 min and washed prior to analysis. Samples were collected with Miltenyi MCSQuant10 (Bergisch Gladbach, Germany) and analyzed with FlowJo Software. Secondary antibody in the control experiment was identical to that described supra.

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).