Irdye 800 conjugated secondary antibody

HEK293 cells in 12-well plate were transfected with 1 μg of above expression construct (the V5-epitope tagged version) using 2 μL of Lipofectamine 2000 under OPTI-MEM medium (Thermo Fisher Scientific) for 24 h. The transfected cells were washed once with PBS, and then lysed with 200 μL of SDS-PAGE sample buffer. The cell lysates were sonicated and heated at 60°C for 5 min. The proteins were separated by 8% SDS-PAGE and transferred to a NC membrane (PALL, 66485) by Pierce 2 Fast Blotter (Thermo Fisher Scientific, B103602038). The membrane was first blocked with 5% BSA/TBST (10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) for 40 min, and then incubated with rabbit anti-V5-epitope antibody (SIGMA) (1:1,000) for 2 h at room temperature. The membrane was washed with TBST for five times (5 min each), and then incubated with IRDye800-conjugated secondary antibody (LI-COR) (1:10,000) for 1.5 h at room temperature under dark and washed as above. The fluorescent signal was detected by Odyssey (LI-COR).

For analysis of chemerin levels in colon explant cultures via SDS-PAGE, 10 ng of recombinant mouse chemerin (aa17–156; R&D) or 30 μL of culture supernatant was boiled with 6× SDS-PAGE loading buffer containing 2-mercaptoethanol. Proteins were separated on a 15% gel and transferred to nitrocellulose membrane (BioRad Laboratories, Mississauga, ON). Blots were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) and then incubated with antichemerin (R&D) overnight at 4°C followed by incubation with an IRDye800-conjugated secondary antibody (LI-COR). Immunoreactive bands were detected by scanning at 800 nm using the Odyssey Infrared System (LI-COR Biosciences, Lincoln, NE).

BALB/cASlac-nu/nu nude mice bearing PDECX1T0326 tumors were orally administered with theliatinib (15 mg/kg) or gefitinib (20 mg/kg). After 8 h, the animals were sacrificed and tumors were harvested. The tumors were homogenized in cold lysis buffer (Cell Signaling Technology, 9803) containing 1 mM PMSF (BIO BASIC INC., PB0425) and after centrifuging the lysates, the protein supernatant (containing 100 μg protein) were mixed with 5× SDS loading buffer and boiled at 100°C for 10 minutes and SDS-PAGE was performed (5% stacking gel at 80V for 20 minutes, then changed to 120V on 10% separating gel for 1h). Proteins were then transferred to nitrocellulose membranes (0.35A for 90 minutes), and incubated with the following antibodies individually: Phospho-EGFR (Tyr1068) (Invitrogen, 44788G), EGFR (Cell Signaling Technology, 2232), phospho-AKT (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 9272), phospho-ERK (Thr202/Thr204) (Cell Signaling Technology, 4370), ERK (Cell Signaling Technology, 4695). After washing with the 1X TBST, the blots were incubated with the secondary IRDye 800-conjugated secondary antibody (LI-COR, 926-32211) and detected with chemiluminescence system.

Total proteins were extracted with RIPA buffer (Beyotime, Zhejiang, China) containing protease inhibitor cocktail tablets (Roche Applied Science, Mannheim, Germany), and separated by SDS‐PAGE (Invitrogen). Then they were transferred onto PVDF membranes and probed with antibodies against Nav1.5, Kv4.3, and Cav1.2 (ASC‐005, APC‐017, and ACC‐003, Alomone Labs, Jerusalem, Israel), Kir2.1 and Kv1.4 (19965‐1‐AP, and 19697‐1‐AP, ProteinTech Group, Wuhan, Hubei, China), Nup107 and GFP (ab73290, and ab290, Abcam, Cambridge, MA, USA), β‐actin, and GAPDH (66009‐1‐Ig and 60004‐1‐Ig, ProteinTech Group). After washing, blots were treated with the appropriate IRDye 800 conjugated secondary antibody (072‐07‐15‐06 and 072‐07‐18‐06, LI‐COR Biosciences, Lincoln, NE, USA) at room temperature for 1 hours. Images were recorded using the Odyssey infrared imaging system and analysed using the Odyssey Application Software v2 (LI‐COR Biosciences).