Semi dry transfer system

The EV-A71-infected and gemcitabine-treated RD cells were incubated for 8 h before total cellular proteins were extracted using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, #78501) containing protease inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific, #78440) and EDTA. Proteins were separated in 10% polyacrylamide gel and transferred to nitrocellulose membranes using the Bio-Rad semidry transfer system. Mouse anti-EV-A71 VP2 protein (Millipore, #MAB979) was used to detect EV-A71 VP0 and VP2 proteins, mouse anti-EV-A71 3D protein (GeneTex, #GTX630193) was used to detect EV-A71 3D and 3CD proteins and mouse anti-actin (Millipore, #MAB1501) was used as loading control. Goat anti-mouse IgG conjugated to HRP (Thermo Fisher Scientific, #31430) was used as the secondary antibody.

Cells were seeded in 6-well plates at 2 × 10⁴ cells/well and starved for 24 h, and then pre-treated with drugs for 2 h before 5-HT stimulation. After 4 h the cells were harvested and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. Protein extracts were divided equally, separated by 12% SDS-PAGE, and then transferred to nitrocellulose membranes via Bio-Rad semi-dry transfer system. The membranes were blocked in 5% non-fat dried milk for 1 h and then sequentially incubated with the primary antibodies overnight at 4 °C and the DyLight 800 AffiniPure goat anti-rabbit IgG or DyLight 680 AffiniPure goat anti-mouse IgG secondary antibodies (EarthOx, CA, USA) for 2 h at room temperature. Lastly, ODYSSEY Infrared Imaging System (LI-ORBiosciences, Lincoln, NE, USA) visually modelled and quantified the immunoreactive bands.

HM, NM, pCMV‐Vector and pCMV‐FAM83D cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (20 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGTA, 1% NP‐40, 1% sodium, deoxycholate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β‐glycerophosphate, 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin and 1 mmol/L PMSF). Total proteins (20‐30 µg) were separated through polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using a semi‐dry transfer system (Bio‐Rad). The membrane was incubated at 4°C overnight with specific primary antibodies for FAM83D (Biorbyt, orb183501), AKT (Cell Signal Technology, CST, MA, cat#: 4691), p‐AKT (CST, MA, cat#:4060), ERK1/2 (CST, MA, cat#:9102), p‐ERK1/2 (CST, MA, cat#: 9101), c‐Raf (CST, MA, cat#: 9422), p‐c‐Raf (CST, MA, cat#: 9421), EGFR (CST, MA, cat#: 4267), p‐EGFR (CST, MA, cat#:2234), P38 (CST, MA, cat#:9212), p‐P38(CST, MA, cat#:9211), N‐cadherin (CST, MA, cat#:13116), ZO1 (CST, MA, cat#:8193) and beta‐actin (CST, MA, cat#: 4970) used at a dilution of 1:1000. Subsequently, the membrane was incubated with secondary antibody (Bio‐Rad, CA, cat#: 1706515 in 1:5000 dilution) for 1 hour at room temperature. Clarity™ Western ECL Substrate (Bio‐Rad, cat#: 1705060) was used for the detection of protein signals. The signals were captured using the ChemiDoc™ XRS + Imaging Systems (Bio‐Rad, CA).

Tumor samples were processed as described earlier [54 (link)]. Preparation of cell lysates, SDS-PAGE and immunoblotting were performed as described elsewhere [55 (link)]. In brief, 40–80 µg protein per lane was transferred to PVDF membranes using a semi-dry transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked for 60–90 min with non-fat dry milk powder (5%, w/v) in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBS-T). For detection of Sig1R, membranes were incubated with primary antibodies in bovine serum albumin (BSA, 2% w/v) in TBS-T (PA5-30372, 1:500, Thermo Fisher Scientific (Waltham, MA, USA), (tumor samples), respectively, ab53852, 1:200, Abcam (Cambridge, UK) (cell lysates)), followed by incubation with peroxidase-conjugated secondary antibody (anti-rabbit IgG, A0545, 1:5000, Sigma-Aldrich, Steinheim, Germany). Proteins were visualized using Super Signal West Pico/Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and a CELVIN^®S Chemiluminescence Imaging system (Biostep, Burkhardtsdorf, Germany). For detection of loading control, membranes were stripped and further processed using mouse anti-β-actin antibody (A5316, 1:1000, Sigma-Aldrich) and anti-mouse IgG (A9044, 1:10,000, Sigma-Aldrich) as described elsewhere [54 (link)].

Whole cell lysates were prepared using Laemmli buffer, and aliquots of protein were separated on 10% polyacrylamide gels by electrophoresis (SDS-PAGE), as described previously [19 (link),25 (link)]. The proteins were transferred to nitrocellulose membranes using a semi-dry transfer system (Bio-Rad, Hercules, CA, USA), blocked in 5% non-fat dry milk solution, and incubated overnight at 4 °C with the primary antibodies as indicated. The next day, blots were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h and visualized with LumiGLO^® (KPL, Gaithersburg, MD, USA). The densitometric analysis was performed using Labworks Software (UVP, Cambridge, UK). Antibody details are provided in Supplementary Materials Table S1.

Western blotting analysis was performed as previously reported [28 (link)]. Whole-cell lysates were used to extract proteins from rat brain tissues in radioimmunoprecipitation assay (RIPA) lysis buffer and were quantified for protein concentration using a bicinchoninic acid (BCA) kit. The protein sample was mixed with the loading buffer and heated in a boiling water bath for denaturation. The same concentration of denatured protein sample was added to each loading well. Proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes using a semidry transfer system (Bio–Rad, USA). After blocking in defatted milk powder for 1 h at room temperature, the membranes were washed with phosphate buffer solution with Tween 20 (PBST) and incubated overnight at 4°C with primary antibodies against FBXW7, HIF-1α VEGF and GAPDH (all purchased from Abcam). Next, the membranes were washed with PBST (5 times × 3 min) and incubated with HRP-secondary antibodies for 1 h. Blots of target proteins were developed using an enhanced chemiluminescence (ECL) kit. Using GAPDH as the loading control, the relative expression of target proteins is expressed as the gray value ratio of the target protein to GAPDH.

According to the method described by Tian et al. (2020) [45 (link)], the WB procedure was as follows. Colon tissue was ground in the presence of liquid nitrogen and then lysed in RIPA lysis buffer for 30 min on ice. The lysates were centrifuged under the condition of 12,000 rpm at 4 °C for 10 min. The protein concentrations of the supernatants were detected by using a BCA protein assay kit. Then, the same amount of protein was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Hybond, Sunnyvale, CA, USA) using a semidry transfer system (Bio-Rad, Hercules, CA, USA).The membranes were incubated with specific antibodies against β-actin, ZO-1,occluding, and claudin-1 overnight at 4 °C, and then HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. All of the antibodies were diluted in tris buffered saline (TBS). The protein signals were analyzed using an ECL detection system (Tanon, Nanjing, China).