Anti p fak antibody

Human HIF‐1α, Kindlin‐2 plasmid, HIF‐1α siRNA, and Kindlin‐2 siRNA were generated by HanBio Technology (Shanghai, China). Specific siRNAs targeting human HIF‐1α (HIF‐1α siRNA) and Kindlin‐2 (K2 siRNA) were designed and synthesized by Biomics Biotechnologies Co., Ltd (Nantong, China). HIF‐1α antibody (Affinity, BF0593, USA), anti–Kindlin‐2 antibody (Sigma, K3269, USA), anti–p‐FAK antibody (Abcam, ab4792, UK), and anti–P4HA1 antibody (Proteintech, 12658‐1‐AP, China) were used in this study.

Doxycycline-inducible cells were seeded at a density of 4×104 in glass bottom plates (Greiner Bio-One International AG, Frickenhausen, Germany) and cultured with 500 ng/ml of doxycycline for 3 days. Cells were washed with 1X PBS three times and fixed with 4% PFA for 15 min at room temperature. Cells were then permeabilized for 10 min with 0.25% Triton X-100 in PBS at room temperature. Non-specific binding was blocked by 0.3% goat serum in PBS for 1 hr at room temperature. Subsequently, for FLAG-Cdc42 expressing cells and Cdc42 expressing inducible cells, cells were incubated with anti-FLAG antibody (1:1000; SIGMA), anti-IQGAP1 antibody (1ug/ml; Abcam), and for U87MG CA-inducible cells, anti-pFAK antibody (5ug/ml; Abcam) at 4°C overnight, respectively. After washing with PBS three times, cells were incubated with Alexa Fluor 488-conjugated secondary antibody (1:500; Molecular Probes) or Phalloidin 594 (1:500, Molecular Probes) together with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) for 1h at room temperature. Fluorescence signal was visualized and photographs were taken using a Zeiss Axiovert 200M Spinning Disc confocal microscope (Carl Zeiss Canada, Toronto, ON, Canada) equipped with a Hamamatsu Back-Thinned EM-CCD camera (Hamamatsu Corporation, Bridgewater, NJ).