Anti pfak y397

Antibodies used were as follows: anti-Paxillin (RRID:AB_647289), anti-GM130 (RRID:AB_398141), anti-p130Cas (RRID:AB_397667) and anti-RACK1 (RRID:AB_397577) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID:AB_2122095) (Abcam, Cambridge, UK), anti-CoxIV (RRID:AB_10694213), anti-FAK (RRID:AB_10694068), anti-pFAK Y397 (RRID:AB_2173659), anti-pPaxillin Y118 (RRID:AB_2174466), anti-Rab7 (RRID:AB_1904103), anti-pSrc Y416 (RRID:AB_331697), anti-Src (clone 36D10) (RRID:AB_10693939), anti-LC3B (RRID:AB_2137707) and anti-GAPDH (RRID:AB_10622025) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID:AB_10842517), anti-Ambra1 (RRID:AB_2636939) and anti-pSrc Y416 (RRID:AB_309898) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID:AB_310789) (Millipore, Billerica, MA, USA). LC3B antibody was from MBL (RRID:AB_1279144) (MBL International, Woburn, MA, USA). TRITC-phalloidin was purchased from Life Technologies (RRID:AB_2572408) (Paisley, UK). Anti-rabbit (RRID:AB_2099233) or mouse (RRID:AB_330924) peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ, USA) and PF562271 from Pfizer (Groton, CT, USA).

Whole-cell lysates were prepared using pre-chilled RIPA (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate). The cell lysates were centrifuged at 12,000×g for 20 min at 4°C and the supernatants were collected for protein concentration determination. The total proteins were separated on 10% SDSPAGE gel and blotted onto a nitrocellulose membrane (Millipore). The membrane was incubated with blocking buffer and then was incubated overnight with appropriate primary antibodies at 4 °C. The primary antibodies used were anti-TSP1 (A6.1; Santa Cruz, USA), anti-p-FAK (Y397) (Cell Signaling Technology, USA), anti-FAK (Cell Signaling Technology, USA), anti-MMP2 (Cell Signaling Technology, USA), anti-MMP9 (Cell Signaling Technology, USA), anti-Fibronectin 1(Abcam, Cambridge, UK, USA), and anti-GAPDH (Santa Cruz, USA). Membranes were then washed three times in TBST solution for 15 min each time, and then incubated with secondary antibodies. The membranes were visualized using LI-COR infrared imaging system (LI-COR) following the manufacturer's guidance.

Antibodies used were the following: anti-Glu-tubulin (Abcam, cat# ab48389, 1/250 dilution for IF, and 1/1000 for WB); anti-α-tubulin (Sigma-Aldrich, cat# T6199, 1/1000 dilution); anti-actin (Ab-5, BD Biosciences, cat# 612656, 1/4000 dilution); anti-FAK (BD Biosciences, cat# 610088, 1/250 dilution); anti-pFAK(Y397) (Cell Signaling Technology, cat# 3283 S, 1/250 dilution); anti-phospho-myosin light chain 2 (Ser 19) (Cell Signaling Technology, cat# 3671, 1/200 dilution); anti-myosin (clone MY-21, Sigma cat# M4401, 1/250 dilution); anti-TTL (Proteintech cat# 13618-AP, 1/500 dilution). Images of uncropped WBs are shown in Supplementary Fig. 10.
For IF, cells were plated onto gels and were fixed with 4%PFA in PBS (phosphate-buffered saline) for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked with 3% BSA for 1 h, and incubated with primary antibodies overnight at 4 °C. Secondary antibodies were conjugated with Alexa 488, 546, or 647 (Alexa Fluora series from Thermo Fisher Scientific). Nuclei or whole-cell area was stained with DAPI or cell mask blue stain, respectively. For determination of percentage of cells with a Glu-MT network, positive cells were defined as those having  > 10–15 distinctly stained Glu-MT fibers.