Clic4

Cell or EV extracts were denatured in Laemmli buffer and incubated at 95°C for 10 min. 10 μg of protein extract (for cell lysates) or equal number of EVs (8.50 × 10⁸ EVs per lane, measured by NTA) were loaded on 4–20% polyacrylamide gels (Bio-Rad Laboratories, Inc). The following antibodies were used: CD9 (Rat, 553758; BD Biosciences), RalA (mouse, 610221; BD Biosciences), RalB (mouse, 04037; Millipore), Glypican 4 (Rabbit, PA5-97801; Thermo Fisher Scientific), antibodies specifically recognizing the short and long isoforms of CD146 were previously described (Kebir et al., 2010 (link)), Clic4 (mouse, 135739; Santa Cruz Biotechnology), α-tubulin (mouse, CP06; Millipore) and Secondary horseradish peroxidase-linked antibodies: anti-Rat (GE healthcare; NA935), anti-Mouse (GE healthcare; NA 931) and anti-rabbit (GE healthcare; NA934). Acquisitions were performed using a PXi system (Syngene). Intensities were measured using the Fiji software.

Total proteins were extracted from the fibroblasts in RIPA buffer and resolved by SDS-PAGE (10–15% Tris-Glycine). The proteins were transferred onto nitrocellulose membranes (Amersham biosciences) and probed with antibodies that were specific for α-SMA (Abcam), CLIC4 (Santa Cruz), GLI1 (Santa Cruz), GLI2 (R&D systems), SMAD3, β-catenin, c-Jun (Cell signalling), and β-Actin (Sigma). The immunoblots were visualized with species-specific HRP conjugated secondary antibodies (Sigma) and ECL (Thermo/Pierce) on a Biorad ChemiDoc imaging system. Densitometry analysis of the blots was performed using the ImageJ software.

HUVECs and EPCs were directly lysed in 1.25 × loading buffer (62.5 mM Tris–HCl, 2.5% SDS, 12.5% Glycerol, 0.05% bromophenol blue, 125 mM DTT) with Protease Inhibitor Cocktail (11,697,498,001, Roche). Lysates were loaded onto SDS-PAGE gels and after electrophoresis transferred to 0.45 μm PVDF membrane (Millipore). After blocking (5% non-fat dried milk in Tris buffered saline (TBS) with 0.1% Tween-20), membranes were probed with first antibodies overnight at 4 °C. The membranes were then washed by TBST for 10 min × 3 times, incubated with second antibodies at room temperature for 1 h. The chemiluminescence (ECL) system and exposure to film was used to visualize the bands, taking number of different exposures.
Antibodies: CCM3/PDCD10 (Abcam, ab180706, 1:1000); OXPHOS (Abcam, ab110413, 1:1000); S100A11 (Proteintech, 10,237-1-AP, 1:1000); ATPIF1 (Proteintech, 12,067-1-AP, 1:1000);PARK7 (Santa Cruz, sc-55572, 1:500); CLIC4 (Santa Cruz, sc-135739, 1:500); β-actin (CST, 3700S, 1:2000); VEGFR2 (CST, 2479S, 1:2000); Tie2 (CST, 4224S, 1:500); p-Tie2 (CST, 4226S, 1:500); p-AKT (CST, 4060S, 1:1000); β-catenin (Santa Cruz, sc-7963, 1:2000); KLF4 (CST, 4038S, 1:500); p-Smad2 (CST, 3108S, 1:500); p-MLC2 (CST, 3674S, 1:1000); MFN1 (Abcam, ab57602, 1:1000); OPA1 (Abcam, ab42364, 1:1000); DRP1(Abcam, ab56788, 1:1000); FIS1 (Enzo, 10,121,719, 1:500); GAPDH (CST, 5174S, 1:5000).

Whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes (Amersham). The membranes were then incubated with nonfat dry milk in Tris-buffered saline containing Tween-20 (TBS-T) to block non-immunospecific protein binding, and then with a primary antibody against KRAS (Santa Cruz, Santa Cruz, CA), CLIC4 (Santa Cruz), Smad2/3 (Cell Signaling Technology, Danvers, MA), phosphorylated Smad2/3 (Cell Signaling Technology), caspase-3 (Cell Signaling Technology), or β-actin (Sigma). After washing with TBS-T, the membranes were incubated with animal-matched HRP-conjugated secondary antibodies (Amersham). Immunoreactivity was visualized with an enhanced chemiluminescence system (Amersham).