Gel doc xr

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of all samples was performed using a Mini-Protein Tetra Electrophoresis System (BioRad Laboratories, Hercules, CA, USA) with 12% acrylamide separating gel and 5% acrylamide stacking gel. Samples (7 μL, 8 mg/mL) diluted with SDS-buffer were loaded into each well. The separating gel was conducted at a constant voltage of 120 V and the stacking gel was conducted at a constant voltage of 80 V. After electrophoresis, the gel was stained for protein by Coomassie blue R250 for 30 min. Then the gel was destained by ultrapure water and then observed by Image Lab™ Software, Version 4.0 (Bio-Rad Gel Doc XR+, Bio-Rad Laboratories, Hercules, CA, USA).

Proteins in cells and tissues were extracted with RIPA lysis buffer (Thermo Fisher). Serum proteins were extracted with Serum Protein Extraction Kit (Qcheng Bio, China). Western blot assays were performed according to details previously reported [23 (link)]. the immuno-complexes were detected with ECL Western Blotting Substrate (Thermo Fisher), visualized with BIO-RAD (BIO-RAD Gel Doc XR+, USA). The following antibodies were used (11000): anti-β-actin (Beyotime, AF0003); anti-α-tubulin (Beyotime, AF0001); anti-GAPDH (Beyotime, AF0006); anti-HSP90 (Proteintech, 60,318–1-Ig); anti-HUR (Proteintech, 11,910–1-AP); anti-EIF2S1 (Proteintech, 11,170–1-AP); anti-VEGF (Proteintech, 19,003–1-AP); anti-Calnexin (Abcam, ab92573); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD81 (Proteintech, 66,866–1-Ig); anti-STUB1 (Proteintech, 55,430–1-AP).
IHC, IF and IP was performed as previously described [24 (link)]. IHC was performed with antibodies against HUR and VEGF (Proteintech, 1:200). IF was performed with antibody against CD31 (Proteintech, 11,265–1-AP, 1:200). The images were scanned by Pannoramic SCAN (3DHistech, Hungary). IP was performed with anti-HSP90 antibody (Proteintech, 1:200) and appropriate control IgG (Merck Millipore), and the immunoprecipitate was then collected by centrifugation and analysed by SDS-PAGE.

(i) DNA preparation. To validate the assay, we selected an additional 38 AzmR (Salmonella Typhi, n = 32; Salmonella Paratyphi A, n = 06) and 62 randomly selected non-AzmR (Salmonella Typhi, n = 48; Salmonella Paratyphi A, n = 14) isolates isolated between 2016 and 2018 in Bangladesh. All the isolates (n = 113, including 13 from Hooda et al. [14 (link)]) selected for validation (see Table S1) were subjected to cell lysis by a simple boiling method. In brief, a single colony from MacConkey agar (Oxoid, Thermo Scientific) was suspended in a 1.5-ml microcentrifuge tube containing 200 μl of sterile DNase-free water. The tube was placed in a heating block at 100°C for 20 min and then centrifuged at 12,000 rpm for 5 min. The supernatant was used for downstream applications.
(ii) Duplex PCR. The total volume of the duplex PCR assay was 25 μl, with 5× master mix (Hot FIREPol; Solis BioDyne, Tartu, Estonia). The final concentrations of the primers were 0.1 and 0.8 μM for the parC and acrB genes, respectively. Amplification was performed in a thermal cycler (ProFlex 3 × 32; Thermo Scientific) (Fig. 5A). The thermocycling conditions are shown in Fig. 5A. Amplified PCR products were run on 2% agarose gel (Invitrogen, Carlsbad, CA) at 100 V for 60 min and visualized on a Bio-Rad Gel Doc XR+ (Bio-Rad, Richmond, CA).

The plasmid-transfected cells were harvested in cell lysis buffer, supplemented with 1% PMSF (Beyotime, Shanghai, China). The cell lysates were resolved on 12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Merck, Darmstadt, Germany). The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.2% Tween-20 (TBST) at RT, followed by incubation with anti-poCD69 mAb supernatant or anti-6×His tag mAb for 1 h at RT. After washing 5 times, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) antibody (Bioworld Technology, Dublin, OH, USA) was added and incubated for another 1 h at RT. After washing 5 times, the specific bands were developed using enhanced chemiluminescence (ECL) reagents, NcmECL Ultra (NCM Biotech, Suzhou, China), and visualized on Bio-Rad GelDoc XR+ (Bio-Rad, Hercules, CA, USA).

Tissue samples and PBMCs were lysed using 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) and protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Lysate was centrifuged at 6000 × g for 10 minutes at 4°C and supernatant was collected for protein determination using a BCA protein assay kit (ThermoFisher). A 50 µg protein sample was resolved by SDS gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were probed using primary antibodies for adenylate kinase 2 (AK2), creatine kinase muscle (CKM), pyruvate kinase liver and red blood cell (PKLR), pyruvate kinase muscle (PKM) (all kinase antibodies from Fisher Scientific), GAPDH (Cell Signaling) or β‐actin (Cell Signaling). Visualization was performed using SuperSignal West Dura or Femto ECL Substrate (ThermoFisher) and a BioRad Gel Doc XR (BioRad).

After cells treated with/without 100 ng/ml rhVEGF, 100 nM apatinib or 100 ng/ml rhVEGF + 100 nM apatinib, cells were lysed using lysis buffer (Cell Signaling Technology, Danvers, USA) to extract total protein. Protein lysates were separated by 10% SDS-PAGE, followed by transfer to nitrocellulose membranes. The membrane was then blocked with 5% milk diluted in PBS at room temperature for 1 h, followed by incubated with 1:1000 VEGFR2 antibody (ab10972, Abcam, Cambridge, MA, USA),1:5000 p-VEGFR2 (ab38473, Abcam), 1:2000 p-MEK (2338, CST), 1:1000 MEK (4694, CST), 1:2000 p-ERK1/2 (4370, CST), 1:1000 ERK (4695, CST), 1:2000 slug (ab51772, Abcam), 1:3000 Snail (ab53519, Abcam), 1:2500 MMP9 (ab38898, Abcam), 1:1500 P-AKT (ab81283, Abcam), 1:1500 AKT (ab179463, Abcam) and 1:5000 GAPDH antibody (ab8245, Abcam) overnight at 4 °C separately. Once primary antibodies were washed, membrane was incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Sangon Biotech, Shanghai, China). Protein bands were detected by incubating the membrane with Western Bright enhanced chemiluminescence working solution (Advansta, Menlo Park, CA, USA). The film (Kodak XBT-1, Carestream, Xiamen, China) was scanned with Bio-rad Gel Doc XR+ (BIO-RAD, Shanghai, China).