Ecl reagent

Protein was extracted from TLR9 siRNA or scrambled RNA transfected HUVECs by Cellytic MT buffer (Sigma) according to the manufacture’s protocol. The concentration of protein was determined using BCA kit (Thermo Scientific). Membranes were incubated with HRP-conjugated secondary antibody (Cell signalling, MA, USA). The signals were developed with ECL reagent (Sigma) and captured by an electronic imaging system (Konica Minolta). β-actin (Cell signalling) was used as house-keeping control. Each samples was loaded as 10 μg protein in 40 μL. The quantification was achieved by normalizing each sample to its β-actin expression.

Protein samples were obtained by using RIPA lysates containing protease inhibitors. Utilizing the Double (BCA) Protein Quantification Kit's guidelines (BCA 1-1 KT; Sigma Aldrich Chemical Company), the protein concentration of every sample was quantified. After that, a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel was used to separate the proteins via electrophoresis, after which, the protein was moved from the glue to a nitrocellulose membrane with a pore diameter of 0.2 μmol/L. In addition, to block non-specific binding sites, a solution of 1 tris buffer salt Tween (TBST) with 5% skim dry milk was added to the membrane, and it was gently shaken for 1 h at room temperature (RT). The membrane was subjected to incubation with the primary antibody and then incubated overnight at 4 C with GAPDH as an internal reference. Secondary antibodies were introduced, and the reaction was performed for 1 h at RT. In addition, an electrochemiluminescence (ECL) reagent (Sigma Aldrich Chemical Company) was employed to visualize the protein bands. The ratio of target and inner reference bands represents protein levels as determined by Image Pro Plus 6.0 (Media controlnetics, San Diego, California), and the internal reference used was GAPDH.

Spore coat proteins were extracted from the purified spores using an SDS-DTT extraction buffer, as described in detail elsewhere [30 ]. For western blot analysis, 50 µg of the extracted proteins were fractionated on 10% SDS-PAGE, electro-transferred to polyvinylidene difluoride (PVDF) membrane (Pall, Hercules, CA, USA) using minitransfer blot (Bio-Rad, Hercules, CA, USA). The Western blot assays were performed using previously described protocols [25 (link)]. Reactive bands were detected with enhanced chemiluminescence (ECL) reagent (Sigma-Aldrich, St.Louis, Missouri, USA) and exposed to X-film as previously described [25 (link)]. For dot blot analysis, 2 µL of 10-fold dilution of the coat proteins with a defined amount was spotted on the FVDF membrane. After treatment, as described in Western blot analysis, the filter was stained with Metal Enhanced DAB Substrate kit (Solarbio, Beijing, China). Images were analyzed densitometrically using Image J.

To evaluate related protein level after treatment, HNSCC cells were collected and lysed by RIPA lysis buffer. Proteins with equal concentration were subjected to the SDS-PAGE and transferred to PVDF membrane (BIO RAD, USA). All samples were blocked by nonfat milk (5%) and incubated with primary antibodies overnight at 4°C. The primary antibodies included anti-EMP1 (Santa Cruz Biotechnology, CA, USA), anti-YAP (Cat, #ab52771), anti-TAZ (Cat, #560235), anti-nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX4) (Cat, #ab133303), anti-acyl-CoA synthetase 4 (ACSL4) (Cat, #ab155282), anti-GPX4 antibody (Cat. #ab125066), anti-Rac1 (Cat. #ab33186), anti-GAPDH (CST, #5176), and anti-nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) (Cat, #ab55831). Antibodies for phospho-epidermal growth factor receptor (EGFR) and EGFR were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-AK, AKT, Phospho-ERK1/2, and ERK1/2 were from Cell Signaling Technology (Beverly, MA). On the next day, all membranes were incubated with the secondary antibody incubation for 2 hours at room temperature. The expression result was detected by ECL reagent (Sigma, USA, WBULS0100). Per condition analyzed a minimum of 10,000 cells.