Protease inhibitor

Proteins were extracted from A549 and H1299 NSCLC cells on ice using RIPA buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitors (Cell Signaling Technology, Inc.) for 20 min. Protein concentration was measured using Bradford Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (15 µg) were separated via SDS-PAGE on a 10% gel and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk dissolved in TBST for 1 h at room temperature and incubated with primary antibodies against E-cadherin (cat. no. ab194982; 1:1,000), N-cadherin (cat. no. ab202030; 1:1,000), vimentin (cat. no. ab193555; 1:1,000), IL-9 (cat. no. ab203386; 1:1,000), STAT3 (cat. no. ab68153; 1:1,000), phospho-STAT3 (cat. no. ab76315; 1:1,000) and β-actin (cat. no. ab179467; 1:5,000), which were all purchased from Abcam, overnight at 4°C. Subsequently, membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. ab150077; 1:10,000) for 1 h at room temperature. Enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology) was used to detect the signal on the membrane. The data were analyzed via densitometry using Bio-Rad Image Lab software (version 4.1; Bio-Rad Laboratories, Inc.) and normalized to the expression of the internal control, β-actin.

Cells were lysed in lysis buffer containing protease inhibitors (Cell Signaling, USA). BCA protein assay reagent (Thermo Fisher Scientific, USA) was used to determine the concentration of the isolated proteins. 25 μg of the protein was fractioned by SDS-PAGE and afterwards transferred to a PVDF membrane (EMD Millipore, USA). The membrane was then incubated with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1 h. The membrane was washed once with TBST, followed by incubation with primary antibodies against MDR1 (Novus Biologicals, USA) (1:1000), α-enolase (Boster, China) (1:1000), XIAP (Abcam, USA) (1 μg/mL), cleaved caspase-3 (R&D systems, USA) (0,5 μg/mL), E-Cadherin (1:500), N-Cadherin (1:1000) (Abcam, USA), and β-actin (1:10,000) (Sigma Aldrich, USA). The membrane was then washed three times for 5 mins with TBST on a shaker and incubated with HRP-conjugated goat anti-mouse (1:10,000) or goat anti-rabbit (1:10,000) IgG secondary antibodies (Abcam, USA) and visualized via Immobilon Western Chemiluminescent HRP substrate (EMD Millipore, USA). Densitometry of blots was analyzed using Image J.

HeLa or MDCK cells transfected with WT or mutant Cx43 cDNAs were trypsinized and grown for 24 h, rinsed in ice-cold PBS, and lysed in lysis buffer (1× RIPA buffer, cat. no. 9806; Cell-Signaling Technology) in the presence of protease inhibitors (cat. no. P8340; Sigma-Aldrich) and 1 mM Na₃VO₄ and 1 mM β-glycerophosphate (to block phosphatase activity) for 20 min on ice. A quantity of 10 μl of Protein-G Dynabeads (cat. no. 10003D; Invitrogen)/sample was washed in 500 μl of PBS/0.02% Tween20. For ZO-1/Cx43 coimmunoprecipitations, the beads were incubated with 1 μl of mouse monoclonal ZO-1 antibody for 1 h at RT. For clathrin heavy-chain/Cx43 coimmunoprecipitation, the beads were incubated with 1 μl of mouse monoclonal clathrin heavy-chain antibody (cat. no. 610499; BD Transduction Laboratories) for 1 h at RT. Bead/antibody mixtures were washed twice in 500 μl PBS/0.02% Tween20 and added to cell lysates, which were first centrifuged at 14,000 rpm to remove cell debris and organelles. Cleared cell lysates (typically 800 µl prepared from a 6-cm-diameter culture dish of confluent cells) were incubated with antibody/beads conjugates for 2 h at RT, washed twice in 500 μl of lysis buffer, and eluted with 30 μl of 2 × SDS–PAGE sample buffer. Beads were boiled for 5 min, and eluted proteins were analyzed by SDS–PAGE and Western blot analyses.

Whole-cell lysates were isolated from sensory neuron cultures using 1× Chaps Cell Extract buffer with protease inhibitors (Cell Signaling Technology). Twenty micrograms of protein was run on 10% Tris-HCl polyacrylamide gels (Bio-Rad), transferred to PVDF membrane (Millipore), and probed following standard methods. Primary antibodies used were rabbit anti-GAPDH (Sigma-Aldrich; G9545), rabbit anti-LRRK2 phospho S935 (Abcam; ab133450), rabbit anti-LRRK2 (Cell Signal; 5559), rabbit anti p62 (Novus; NBP1-48320), rabbit anti-LC3B (Cell Signal; 3868), mouse anti PHF-Tau (Thermo; MN1020), mouse anti-Tau (Cell Signal; 4019), and mouse anti-SNCA (DSHB; H3C-s). Secondary antibodies anti-rabbit IgG HRP (Promega; W4011) and anti-mouse IgG HRP (Promega; W4021) were used.