Precision red

Endogenous Ras-GTP levels were measured using an enzyme-linked immunosorbent
assay-based G-LISA kit (Cytoskeleton, Denver, CO, USA, catalogue #BK131)
following the manufacturer's instructions. Briefly, cells were plated and
allowed to grow to roughly 50% confluence before being washed with PBS
and lysed in 100 μl of ice-cold lysis buffer in the presence of
protease inhibitor cocktail. The lysate was clarified by centrifugation at
10,000 g for 1 min, and snap frozen in liquid nitrogen.
After normalizing protein concentration using PrecisionRed (Cytoskeleton),
samples were added in triplicate to 96-well plates coated with Ras GTP-binding
protein and incubated at 4 °C for 30 min at 300 revolutions
per minute (r.p.m.). After washing, bound Ras-GTP levels were determined by
subsequent incubations with an anti-Ras antibody and a secondary horseradish
peroxidase (HRP)-conjugated antibody, followed by addition to an HRP detection
reagent. Ras activity was quantified by measuring absorbance at 490 nm.
Background was determined with a negative control well loaded with lysis buffer,
and experiments for each cell type were repeated three times.

Cells were grown to 80% confluence in a six-well plate and lysed with radioimmunoprecipitation assay buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Triton X-100, and 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktails (#78438, #78427; Thermo Fisher Scientific). Lysates were spun at maximum speed for 10 min before protein levels were measured using Precision Red (#ADV02; Cytoskeleton). Samples were run using precast 4–12% NuPAGE Bis-Tris Acrylamide Gels (#NP0321; Thermo Fisher Scientific) and transferred to a 0.45-µm nitrocellulose membrane (#10600002; GE Healthcare). Membranes were blocked in 5% BSA in TBS + 0.1% Tween-20 for 30 min before being incubated with specific primary antibodies overnight and the corresponding fluorescently conjugated secondary antibody for 1 h (#A21206 and #A10038, Invitrogen; #SA5-35521 and #SA5-35571, Thermo Fisher Scientific) before being analyzed using Image Studio Lite (LiCor).

Protein lysates were prepared in 1X RIPA buffer from isolated cells (see above). Protein concentration was estimated using Precision Red (Cytoskeleton, Inc.) and NanoDrop One spectrophotometer. Proteins were separated using an 8% polyacrylamide separating/resolving gel, 4% stacking gel, and electrophoresis. Protein was transferred to PVDF membrane, and membrane was blocked with 3% bovine serum albumin in tris-buffered saline with Tween-20, before incubating with primary antibodies. Primary antibody dilutions: rabbit anti-gapdh (1:1,000) (Cell Signaling Technologies); mouse anti-N-cadherin (3B9) (1:1,000) (Invitrogen); rat anti-Pdgfrβ (CD140b) (1:1,000) (Invitrogen); rabbit anti-Rbpj (1:1,000) (Cell Signaling Technologies). HRP-conjugated secondary antibody dilutions: anti-mouse (1:1,000); anti-rabbit (1:1,000); anti-rat (1:1,000) (Cell Signaling Technologies). Chemiluminescent substate was applied to the membrane and bands were detected and quantified with BioRad ChemiDoc XRS+ and ImageLab software.

Melanocytes in a dish were lysed in RIPA buffer (150 mm NaCl, 10 mm Tris pH 7.5, 1 mm EDTA, 1% Triton X‐100, 0.1% SDS) with Halt protease inhibitor (Pearce, Thermo Fisher, Loughborough, UK) for 10 min on ice. Samples were spun at 20 000g for 10 min at 4°C, and pellets were discarded. Equal protein, measured using Precision Red (Cytoskeleton, Cambridge, UK), was separated by SDS‐PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% BSA PBS‐T for 1 h at room temperature, incubated on a rocker overnight at 4°C with 1/1000 rabbit anti‐WASH (Millipore ABS72), 1/500 rabbit anti‐SWIP (Proteintech 51101‐1‐AP, Manchester, UK), 1/500 rabbit anti‐Fam21C (Millipore ABT79), 1/500 rabbit anti‐CCDC53 (Millipore ABT69), 1/2000 rabbit antistrumpellin (Abcam ab101222) or 1/2000 rabbit anti‐FKBP135 (WAFL) (Abcam ab14432) and 1/20 000 mouse anti‐alpha‐tubulin (Sigma T6199) loading control in 5% BSA PBS‐T. Blots were washed in PBS‐T and then incubated with 1/10 000 goat anti‐mouse Alexa 680 and 1/10 000 goat anti‐rabbit Dylight^™ 800 4× PEG (Life Technologies) in 5% BSA PBS‐T. Blots were imaged on a Li‐Cor ODYSSEY^® CLx. Protein band integrated density analysis was performed using image studio lite software (LI‐COR, https://www.licor.com/bio/products/software/image_studio_lite/) and normalized to 100% in control cells.

SMCs were extracted in HEPES buffer containing 1% Triton X-100 [16 (link)]. Protein was measured using Precision-Red (Cytoskeleton) and equal amounts were then subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. Blots were developed with ECL (GE Healthcare). The following antibodies were used: anti-β-tubulin (Cell Signaling #2146), anti-smooth muscle alpha-actin (Sigma #A2547), anti-VSVG (Roche #11667351001), anti-Rac1 (BD Biosciences, #610650) and anti-RhoA (Santa Cruz sc-418). To analyze MAPK phosphorylation, serum-starved GFP-controls and SMA-SMCs were stimulated with 10% FBS and harvested at the time points indicated. Cell extracts were subjected to Western blot analysis and blots probed with antibodies to recognize the phosphorylated forms of p44/42 ERK, p38 MAPK and p54/46 JNK. All antibodies were from Cell Signaling and used at the following dilutions: #9101 (1:10,000), #9215 (1:1,000) and #9251 (1:500). To control for equal loading, blots were reprobed with HSC70 antibody (Santa Cruz, sc-7298, 1:6000).