Na934v

Cells were lysed with lysis buffer (a cocktail of 20 mM Tris-HCl pH 8.0, 60 mM NaCl, 0.2% glycerol, 0.02% NP-40, 0.04 mM EDTA, 1 mM PMSF, and 1.52 mg/mL protease inhibitor). Proteins were analyzed with SDS-PAGE gel and transferred onto a polyvinyl difluoride membrane (PVDF) (Millipore, Temecula, CA, USA). The PVDF membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies used were anti-Kla (PTM BIO, PTM-1401,1:1000), anti-β-Tubulin (Abmart, M20005H, 1:5000, Shanghai, China), anti-Ldha (Cell Signaling Technology, 2012S, 1:1000, Danvers, MA, USA), anti-Nanog (Bethyl Laboratories, A300-397A, 1:2000), anti-Oct4 (Santa Cruz Biotechnology, sc-5279, 1:1000, Dallas, TX, USA), anti-Esrrb (Perseus Proteomics, H6705-00, 1:1000, Tokyo, Japan), and anti-FLAG (Sigma-Aldrich, F1804, 1:3000). After washing with TBS 3 times, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. The secondary antibodies used were donkey anti-Rabbit (Cytiva, NA934V, 1:5000, Marlborough, MA, USA), goat anti-Rabbit (Santa Cruz, sc-2354, 1:1000), and sheep anti-Mouse (Cytiva, NA931V, 1:5000, Marlborough, MA, USA). ECL solution (Shanghai Epizyme Biomedical Technology Co., Ltd., Shanghai, China) was added, and signals were detected using an automatic chemiluminescence imaging analysis system (Tanon, Shanghai, China).

Proteins (10 µg) were mixed with 2 × Laemmli buffer supplemented with 2-mercaptoethanol, and denatured for 5 min at 95 °C. Samples were loaded in a 4–20% Mini-PROTEAN TGX Precast Gel (Bio-Rad). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad). Membranes were blocked with TBST (0.2% Tween-20 diluted in Tris-buffered saline) containing 5% Blocking-One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies for anti-TRPA1 (rabbit, Polyclonal, 1:500; NB110-40763, Novus Biologicals, LLC, Centennial, CO, USA), and anti-β-actin antibody (mouse monoclonal, 1:200; sc-69879; Santa Cruz, San Diego, CA, USA). After washing with TBST 4 times for 5 min, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for anti-rabbit IgG or anti-mouse IgG (1:2000, NA934V or NA931V; Cytiva, Marlborough, MA, USA) 2 h at room temperature. The bands were visualized using an image analyzer (Amersham Image Quant 800, Cytiva) after reaction in Western Lightning ELC Pro (PerkinElmer, Waltham, MA, USA) or Immobilon (Millipore, Burlington, MA, USA). The band intensity was quantified using ImageJ and normalized to β-actin expression.

HUVECs were lysed on ice using Laemmli buffer, as previously described [5 (link)]. Briefly, 20 µg of total protein for each sample was mixed with a 6x loading dye buffer and loaded onto 10% SDS denaturing poly-acrylamide gels. After transferring proteins to a PVDF membrane (10600021 Euroclone), the membrane was blocked with 5% fat-dried milk (EMR180001 Euroclone) and incubated with primary polyclonal rabbit anti-TMEM230 (1:2500, 21466-1-AP, Proteintech, Rosemont, IL, USA,) and polyclonal goat anti-Lamin A/C (sc 376248 Santa Cruz Biotechnology, Dallas, TX, USA) used as endogenous control at concentration 1:7500. Donkey anti-rabbit (1:20,000, NA934V, Amersham, Cologno Monzese, Mi, Italy) and sheep anti-mouse (1:10,000, NA931V Amersham) were used as secondary antibodies.

Whole‐cell extracts were prepared in radioimmune precipitation assay (RIPA) buffer (50 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1% NP‐40, 0.1% SDS, 0.5% sodium deoxycholate, 0.5 mmol/L DTT, 0.5 mmol/L PMSF and 2.5 mmol/L Roche protease inhibitor cocktail). The extracts were subjected to Western blot analysis as described previously.¹¹ Briefly, 20 to 40 μg of total protein were separated using 7‐12% SDS‐PAGE and transferred to PVDF membrane. The membrane containing transferred proteins was blocked by incubating with 5% BSA containing 0.05% Tween‐20 and incubated overnight at 4°C with primary antibody to detect CYCLIN D1 (Abcam, ab134175), c‐MYC (Abcam, ab62928), SURVIVIN (Abcam, ab76424), α‐TUBULIN (Abcam, ab4074), DKK1 (Abcam, ab109416), DKK3 (Abcam, ab186409), β‐ACTIN (Cell Signaling Technology, 4970), CYCLIN D3 (Cell Signaling Technology, DCS22) and PRMT5 (Thermo Fisher, MA1‐25470). After incubation with primary antibody, the membrane was treated with HRP‐conjugated goat anti‐mouse (Amersham Biosciences, NA931) or anti‐rabbit (Amersham Biosciences, NA934V) secondary antibody. Next, proteins were visualized using the ECL detection kit (Amersham, RPN2209) in a Western blot imager (Flurochem E system, proteinsimple).

Cells were lysed with RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5) containing protease inhibitors. The protein concentration was determined using DC protein assay (Bio-Rad, Hercules, CA, USA), and equal amounts of protein were separated on 6–12% SDS-PAGE Gels and transferred to PVDF membrane (Invitrogen, Waltham, MA, USA). Nonspecific protein binding was blocked using either 3% Bovine Serum Albumin (BSA) or 5% powdered milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h followed by incubation with primary antibodies diluted in the blocking solution, overnight at 4 °C. The following morning, the membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, NA931V, and anti-rabbit, NA934V, Amersham) for 1 h at room temperature. Membranes were then washed and imaged. Images were developed using the Immobilon Western Kit (Millipore, Burlington, MA, USA) and detected on a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA), and Bio-rad (version S.0.2.3.0, Rochester, NY, USA) was used to quantify protein content. GAPDH (1:10,000, no. ab9484, Abcam, Cambridge, United Kingdom) was used as a loading control. (Table 2).

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.