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.
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.