Precision red advanced protein assay reagent

Cytokine levels for IL-1α, IL-1β, IL-6, IL-10, and TNF-α from the F0–F3 generations were quantified simultaneously. Analyses were performed using Bio-Plex 200 suspension array system and Bio-Plex 200 software, version 6.0 (Bio-Rad Laboratories, Mississauga, ON, Canada). We used the MILLIPLEX MAP Rat Cytokine/Chemokine Magnetic Bead Panel (RECYMAG-65K), a precustomized magnetic-bead-based multiplex assay (Millipore Sigma, Burlington, MA, USA) and followed the manufacturer’s protocol. In brief, uterine horns (3 mm) were weighted and diluted with 1x phosphate-buffered saline (PBS) to a concentration of 0.1 mg/mL. Tissues were homogenized using Tissue Lyzer II (Qiagen, Toronto, ON, Canada) with 7 mm stainless steel beads 4 times for 2 min, 25 Hz cycles. Tissue homogenate protein concentrations were quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) with Precision red advanced protein assay reagent (Cytoskeleton Inc., Denver, CO, USA), and then immediately stored at −80 °C until use. Multiplex assay was calibrated and validated before sample analyses. Reagents’ preparation and assay were conducted following the manufacturer’s protocol.

RPE cells were transfected with pEGFP-myosin VI containing various tail truncations or mutations using Lipofectamine 2000 (Invitrogen) and fractionated using the following method (modification of the G-actin:F-actin assay kit from Cytoskeleton): cells were homogenised using a 25-G needle in 250 µl of F-actin stabilisation buffer [50 mM PIPES, pH 6.9, 50 mM NaCl, 5 mM MgCl₂, 5 mM EGTA, 20 mM NaF, 20 mM Na₃VO₄, 5% glycerol, 0.1% NP40, 0.1% Tween 20, 0.1% Triton X-100, 0.1% β-mercaptoethanol, phosphatase (Roche PhosSTOP) and protease (Roche cOmplete Mini, EDTA-free) inhibitors] prewarmed to 37°C, centrifuged at 2000 × g for 5 min to remove unbroken cells and the supernatant was centrifuged at 100 000 × g for 1 h at 37°C. Supernatants (G-actin) were stored on ice; pellets (F-actin) were resuspended in prechilled MilliQ water containing 10 µM cytocholasin D for 1 h on ice. Protein concentrations of supernatants and pellets were determined using the Precision Red advanced protein assay reagent (Cytoskeleton). On average, five times the amount of protein was present in the supernatant compared with the pellet fraction.

The samples derived from human keratinocytes and mouse skin tissues were lysed with RIPA lysis buffer (9806, Cell Signaling Technology). Protein concentrations were determined using Precision Red Advanced Protein Assay reagent (ADV02, Cytoskeleton), and equal amounts of total protein were subjected to electrophoresis with 8%–15% SDS-PAGE gels followed by transfer to PVDF membranes (IPVH00010, Merck Millipore, Burlington, Massachusetts, USA). The membranes were then blocked in ImmunoBlock buffer for 1 hour at room temperature followed by overnight incubation at 4°C with primary antibodies according to the manufacturer’s instructions. Labeling of the primary antibodies was detected using sheep anti-rabbit or sheep anti-mouse antibodies conjugated to horseradish peroxidase (NA934 V and NA931 V, respectively; Amersham Biosciences), developed with the Luminata Forte Western horseradish peroxidase substrate (WBLUF0100, Merck Millipore, Billerica, Massachusetts, USA), and then imaged using Fujifilm LAS-4000 Plus. ImageJ was used for quantification of the band intensity in the images.

The samples derived from human keratinocytes were lysed with RIPA lysis buffer (9806, Cell Signaling Technology, Beverly, MA, USA). The protein concentrations were determined using Precision Red Advanced Protein Assay reagent (ADV02, Cytoskeleton, Denver, CO, USA), and equal amounts of total protein were subjected to electrophoresis with 8–15% SDS–PAGE gels followed by transfer to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Merck Minipore, Burlington, MA, USA). The membranes were then blocked in ImmunoBlock buffer for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies according to the manufacturer’s instructions. The labeling of the primary antibodies was detected using sheep anti-rabbit or sheep anti-mouse antibodies conjugated to horseradish peroxidase (NA934 V and NA931 V, respectively (Amersham Biosciences, Piscataway, NJ, USA)), developed with the Luminata Forte Western horseradish peroxidase substrate (WBLUF0100, Merck Millipore, Billerica, MA, USA), and then imaged using Fujifilm LAS-4000 Plus (Fujifilm, Tokyo, Japan). ImageJ was used for quantification of the band intensities in the images. The antibodies used in these studies are listed in Supplementary Table S4.

Cells were lysed using M-PER mammalian protein extraction reagent (Thermo Scientific) containing a protease inhibitors cocktail (Roche). The lysates were centrifuged at 4°C for 20 min at 16000g and supernatants were collected. Total protein concentration was determined using Precision Red advanced protein assay reagent (ADV02, Cytoskeleton). The supernatants were incubated for 5 min at 95°C with NuPAGE^® LDS sample buffer (NP007, Life Technologies). Equal amounts of protein were loaded and resolved using precast gels from Bio-rad (Mini PROTEAN TGX Gel, Cat number 456-9034) and electro-transferred to nitrocellulose membranes (Hybond, GE Healthcare). The blots were blocked with 5% milk powder and incubated with primary antibody according to the manufacturer’s protocol. Secondary antibody conjugated with horseradish peroxidase (HRP) was used for detection using ECL plus western blotting detection reagent (GE Healthcare). To confirm equal protein loading, all the immunoblots were also probed with anti GAPDH antibody. The immunoblots were scanned on the LI-COR Odyssey FC Imaging System and the signal intensity was analyzed by Image Studio™ Lite analysis software (LI-COR).