Kif3a

Antibodies for PAK1, phospho-PAK1 and phospho-Ser/Thr were from Cell Signaling Technology; IFT88, KIF3A, SuFu, c-Myc, Gli1, acetylated-αTubulin (Ac-Tub), Vav2, GAPDH and β-actin antibdies were from Santa Cruz Biotechnology; Ptch1 antibody, Smo antibody and phospho-Vav2 antibody were from Abcam; Gli2 antibody and Rac1 antibody were from Bioss (Beijing, China); Rac2 and Rac3 antibodies were purchased from Huabio (Hangzhou, China); Flag-tag and HA-tag antibodies were from Beyotime (Shanghai, China); GST-tag antibody was from Yeason (Shanghai, China), and antibody anti-Arl13b was from Proteintech. Alexa555- and Alexa-488 conjugated secondary antibodies were from Life Technology. Recombinant mouse Shh protein (N-Shh, C25II, N-Terminus) was from R&D Systems (#464-SH). Cyclopamine, SAG and NSC23766 were purchased from Selleckchem. Cycloheximide and MG132 were from Beyotime (Shanghai, China).

Immunofluorescence was performed on chamber slides (Nalge Nunc International, Naperville, IL). After rinsed in PBS, samples (cells or limb bud tissues) were fixed in ice-cold methanol and permeabilized with 0.1% Triton X-100 in PBS (PBST). After incubation with blocking buffer for 30 min at room temperature, samples were incubated with primary antibodies against Smo (ab236465, abcam), Gli1 (sc-20687, Santa Cruz), pVav2 (ab86695, abcam), Vav2 (sc-271442, Santa Cruz), acetylated-αTubulin (sc-23950, Santa Cruz), KIF3A (sc-376680, Santa Cruz), IFT88 (sc-84318, Santa Cruz), pPAK1 (#2601, Cell Signaling Technology), PAK1 (#2602, Cell Signaling Technology), Arl13b (17711-1-AP, Proteintech) or β-actin (sc-47778, Santa Cruz) overnight at 4 °C. After washing with PBST, samples were further incubated with Alexa 488-conjugated or 555-conjugated secondary antibody (Life Technology). The nuclei were counterstained with 6'-diamidino-2-phenylindole (DAPI), and immunostaining was analyzed by a laser scanning microscope (Zeiss).

The Co-IP test was evaluated in accordance with previously provided instructions [24 (link)]. Transfected HEK293 cells or DRG tissues were lysed in cold Co-IP RIPA buffer. The lysates were centrifuged, and 5% of each supernatant was taken for the input sample. The remaining supernatants were incubated with 5–10 μg EndoA2 (mouse, Santa Cruz, USA, sc-365704), Piezo2 (rabbit, 1:200, Alomone, Israel, APC-090) or His (mouse, Santa Cruz, USA, sc-8036) antibody at 4 °C overnight and then with protein A/G beads (GE Healthcare, UK) at 4 °C for 4 h. The immunoprecipitated samples were denatured and prepared for immunoblotting. Immunoprecipitation was performed with antibodies against Piezo2 (rabbit, 1:1000, Novus, USA, NBP1-78,624; rabbit, 1:200, Alomone, Israel, APC-090), TACAN/Tmem120a (rabbit, 1:1000, Bioss, China, bs-19952R), ASIC2 (rabbit, 1:1000, Bioss, China, bs-4915R), ASIC3 (rabbit, 1:1000, Abcam, USA, ab190638), KIF5B (rabbit, 1:2000, Abcam, USA, ab167429), KIF5A (rabbit, 1:1000, Abcam, USA, ab5628), KIF17 (rabbit, 1:1000, Bioss, China, bs-3527R), KIF3A (goat, 1:100, Santa Cruz, USA, sc-18745), KIF3B (rabbit, 1:1000, Bioss, China, bs-17085R), His-tag (rabbit, 1:1000, Cell Signaling Technology, USA, 12,698) or myc-tag (rabbit, 1:2000, Bioss, China, bs-23166R). The precipitant was washed, denatured and prepared for immunoblotting.