Ab55456

MDCK ZO-1/ZO-2-KD (ZO KD, clone 3B3; Fanning et al., 2012 (link)) and Ecad-GFP CRISPR Caco-2 (Cavanaugh et al., 2020 (link)) cells were cultured in complete medium (high-glucose DMEM [Corning] supplemented with 10% FBS and 20 mM Hepes [Corning]) and maintained at 37°C with 5% CO₂. For imaging, cells were plated on Matrigel (356231; Corning)-coated coverslips. For transfection of GFP-mito-AP4/FP4 constructs (a gift from Dr. Stephanie Gupton, University of North Carolina at Chapel Hill; Bear et al., 2000 (link)), the PolyJet Transfection Reagent (SignaGen Laboratories) was used per manufacturer’s instructions. Calcium switch buffer (CSB) was prepared by adding 2 mM magnesium dichloride to magnesium-calcium–free PBS (Corning). Antibodies and concentrations used for immunostaining are as follows: rabbit anti-nonmuscle myosin IIB-targeted at myosin tail (909902; BioLegend, 1:300), rat anti-Ecad (sc59778; Santa Cruz, clone DECMA-1, 1:300), mouse anti-VASP (VM2771; ECM Biosciences, 1:150), mouse anti-ARP3 (A5979; Sigma-Aldrich, 1:100), mouse anti-nonmuscle myosin IIA (ab55456; Abcam, 1:100), and mouse anti-α-actinin-4 (sc393495; Santa Cruz, 1:100). Secondary antibodies were purchased from Thermo Fisher Scientific. Actin staining using FITC-phalloidin or Alexa Fluor 647–phalloidin (Invitrogen) was done according to manufacturer’s instructions.

Confluent WT keratinocyte primary cultures were lysed in 1% Triton X-100 buffer (40 mM Hepes, pH 7.5, 120 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM sodium pyrophosphate, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, and protease inhibitors including 2 µg/ml antipain, 10 µg/ml aprotinin, 10 µg/ml benzamidine, 1 µg/ml leupeptin, 1 µg/ml chymostatin, and 1 µg/ml pepstatin A), incubated for 15 min on a shaker at 4°C, and spun down at maximum speed (16.1 × 1,000 g) for 10 min at 4°C. Cell lysates were precleared with 30 µl protein G–coupled Sepharose beads (17-0618-01; GE Healthcare) for 30 min at 4°C. The Bradford assay was used to determine the concentration of protein lysates. Mouse anti–myosin IIA antibodies (ab55456; Abcam) or mouse IgG antibodies (sc-2025; Santa Cruz) were added to lysates (1 µg antibody per 1 mg lysates) and incubated for 3 h on a shaker at 4°C. protein G–coupled Sepharose beads (30 µl) were then added for an additional 45-min incubation with the protein lysates/antibody mixture. Bead-bound proteins were eluted by adding 2× SDS sample buffer containing 5% β-mercaptoethanol and analyzed via Western blotting.

5 µg of each bait protein (purified recombinant keratin proteins: e.g., K5, K6, and K17) was electrophoresed on duplicate 10% SDS-PAGE gels. One gel was stained with Coomassie dye to assess protein loading. The other gel was electroblotted to a nitrocellulose membrane (Bio-Rad) with 0.45-µm pore size. The membrane was blocked in 5% milk in TBST for 30 min. After washing briefly with TBST, the membrane was incubated with the target protein, myosin protein (1.5 µg/ml; MY02-A; Cytoskeleton) in 5% BSA in TBST on a shaker at room temperature for 4 h. After several washes in TBST, the membrane was blocked in in 5% milk in TBST for 30 min at room temperature. Primary antibody (mouse anti–myosin IIA; ab55456; Abcam) was diluted (1:1,000) in 5% BSA in TBST and applied to the membrane overnight at 4°C. Membranes were washed three times (5 min each) with TBST before incubation with HRP-conjugated secondary antibody (1:2,000) for 1 h at room temperature. After three TBST washes (5 min each), membrane bound proteins of interest were detected using Amersham ECL Select Western Blotting Detection Reagent and the FluorChem Q imaging system (ProteinSimple).

Cells were washed in ice-cold PBS and lysed in ice-cold lysis buffer (20 mM Tris, 500 mM NaCl, 1 mM EDTA, and 0.5% Triton, pH 7.5) containing Phosphatase Inhibitor Cocktail 2 and Protease Inhibitor Cocktail (Sigma). After centrifugation at 13,000 g for 10 min at 4°C, supernatants were mixed with the NuPAGE sample buffer (Invitrogen) supplemented with 200 mM DTT and heated at 75°C for 5 min. Approximately 2.5 to 5 µg total protein was loaded onto 3–8% NuPAGE TA 1.0 gradient gels (Invitrogen), resolved by SDS-PAGE, and transferred to a 0.45-µm polyvinylidene fluoride membrane in NuPAGE Transfer Buffer at 20 V for 2 h using the X Cell SureLock Electrophoresis and Western Blotting System (Invitrogen). The membrane was blocked for 1 h with 5% dry milk in TBS (Bio-Rad) containing 0.1% Tween-20 (Sigma), incubated with primary antibodies in TBS/Tween-20 for 2 h, washed in TBS/Tween-20, incubated with secondary antibodies for 1 h, and developed with Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Healthcare). The following antibodies were used: mouse monoclonal antibody to NMIIA (ab55456; 1:30,000; Abcam), rabbit polyclonal antibody to NMIIB (3404; 1:1,000; Cell Signaling), mouse monoclonal antibody to α-tubulin (T9026; 1:5,000; Sigma), and horseradish peroxidase–linked enhanced chemiluminescence anti–mouse and anti–rabbit IgG (1:20,000; GE Healthcare).