Ecl chemiluminescence detection system

Whole cell protein extracts were prepared from 4.5×10⁶cells using 50μL lysis buffer containing 20mM Tris(hydroxymethyl)aminomethane (Tris) pH 7.4, 1mM EDTA, 140mM NaCl, 1% NP-40 supplemented with 2mM sodium vanadate and protease inhibitor cocktail (Sigma-Aldrich, San Louis, MO, USA) for 1 hour at 4°C. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of denatured protein were resolved by 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were blocked for 1 hour at room temperature (RT) in 5% milk/TBS-T. Membranes were incubated overnight at 4°C with primary antibodies against ZAP-70, Mcl-1 and Bcl-2 (Santa Cruz Biotechnologies, Dallas, TX, USA), and GAPDH (Abcam, Cambridge, United Kingdom). Immunodetection was done with the corresponding IgG HRP-linked secondary antibodies (Dako North America, Glostrup, Denmark), and the ECL chemiluminescence detection system (GE Healthcare). Chemiluminescent images were acquired with the LAS-4000 system (Buckinghamshire, United Kingdom). Protein expression was subsequently quantified using the Image J 1.46r software (National Institutes of Health).

Spoligotyping was performed as described previously by Kamerbeek et al.12 (link). Detection of the hybridization patterns was carried out using the ECL^® Chemiluminescence Detection System (GE Healthcare^®, Cleveland, OH, USA).
All isolates were typed by 24-loci MIRU-VNTR using the multiplex amplification procedure described by Supply et al.11 (link).
Spoligotyping and MIRU-VNTR profiles were assigned to lineage, clade, shared international type (SIT) and MIRU International Type (MIT) using the SITVIT WEB international database (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE/index.jsp) and/or the SPOTCLUST tool (http://tbinsight.cs.rpi.edu/run_spotclust.html)16 (link)19 (link).
A dendrogram was constructed based on the MIRU-VNTR and spoligotyping data, as appropriated, using the MIRU-VNTRplus international database15 (link). A cluster was defined as group of two or more strains with identical profile. The Hunter-Gaston index of diversity was computed as described previously20 (link).
A minimum spanning tree was also constructed using the MIRU-VNTRplus database to investigate phylogenetic relationships within the sample and identify clonal complexes. A clonal complex was defined as groups of isolates that are within dual-locus variants of each other.
All statistical analyses were conducted using the IBM^© SPSS^© Statistics v.21 (IBM Corporation, Armonk, NY, USA).

Protein samples were boiled for 10 min in Laemmli buffer (10% (w/v) glycerol, 2% SDS, 10% (v/v) β-mercaptoethanol, and 62.5 mm Tris-HCl (pH 6.8)) and separated by SDS-PAGE before transferring onto nitrocellulose membrane. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies (SUMO1, Flag, or HA), followed by a horseradish peroxidase-conjugated secondary antibody. The secondary antibody was detected with an ECL chemiluminescence detection system (GE Healthcare, Stockholm, Sweden).

Tissues were homogenized in RIPA buffer supplemented with protease inhibitor, phosphatase inhibitor cocktail 1 and cocktail 2 (Servicebio, Wuhan, China), and protein were quantified using Bradford method (BIO-RAD). Proteins were electrophoresed on 10–15% SDS-polyacrylamide gels and transferred to 0.45 μm PVDF membranes (Millipore, Ireland) with a wet transfer system (BIO-RAD, USA) for 45 min. Membranes were blocked and incubated with primary antibodies in 3% BSA in Tris-Buffered Saline Tween-20 (TBST) overnight at 4°C. Blots were then rinsed and incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) (Sangon Biotech, Shanghai, China) and exposed on an ECL chemiluminescence detection system (G&E Healthcare, USA). Information about sources of antibodies was listed as following: GAPDH (BOSTER, China), FAS (Santa Cruz, USA), ACC (Santa Cruz, USA), HSL (Cell signaling, USA), Phospho-HSL (Ser 563) (Affinity, China), PPARγ (Santa Cruz, USA), ATGL (Santa Cruz, USA), PLIN1 (BOSTER, China), and CGI-58 (Santa Cruz, USA).

All cells and fresh tissues were lysed in radioimmunoprecipitation (RIPA) buffer (Thermo Fisher, USA) supplemented with 1% proteinase inhibitor. We performed nuclear and cytoplasmic fraction analyses according to a standard protocol (Thermo Fisher Scientific, USA). Equal quantities of protein were loaded on a 10% polyacrylamide gel. Proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The PVDF membranes were incubated with primary antibodies at 4°C and secondary antibodies at room temperature. Signals were detected using an ECL chemiluminescence detection system (GE Healthcare Life Sciences and Millipore). The following antibodies were used: β-catenin (CST, 8480S, 1:1000, USA), NICD (CST, ab8925, 1:1000, USA), anti-SFRP1 (Abcam, ab4193, 1:1000, USA), anti-DKK3 (Abcam, ab2459, 1:2000, USA), anti-GSK3β (Abcam, ab32391, 1:2000, USA), anti-RUNX3 (CST, 9647, 1:1000, USA), NUMB (Abcam, ab4147, 1:800, USA), anti-GAPDH (CST, 8884, 1:1000), anti-HISTH3 (ABclonal, A2348, 1:1000), goat anti-mouse IgG-horseradish peroxidase (CST, 7076S, 1:3000, USA), goat anti-rabbit IgG-horseradish peroxidase (CST, 7074S, 1:3000, USA) and peroxidase-conjugated affinity-purified bovine anti-goat IgG (Jackson ImmunoResearch, 805-035-180, 1:5000, USA).

NP40 cell lysates (1% NP40, 10% glycerol, 0.05 M Tris pH 7.5, 0.15 M NaCl, 1 mM PMSF, protease and phosphatase Inhibitor cocktails) (Roche and Thermo Scientific, respectively) were resuspended in Laemmli's 2x buffer, separated on SDS/PAGE and blotted onto nitrocellulose membrane (GE Healthcare, Little Chalfont, United Kingdom). Blots were incubated with the following antibodies (Abs): P-Y^694/699-STAT5, catalase (Cell Signaling Technology, Danvers, United States), STAT5 (BD Transduction Laboratories, Franklin Lakes, United States), actin (Santa Cruz, Dallas, United States), Flag M2 (Stratagene, Santa Clara, United States), Glutaredoxin1 (Glrx1) (R&D, Minneapolis, United States). Membranes were developed with the ECL chemiluminescence detection system (GE Healthcare, Little Chalfont, United Kingdom) using specific peroxidase (HRP) conjugated to rabbit or mouse IgG antibodies (Cell signaling Technology, Danvers, United States) or goat IgG (Santa Cruz, Dallas, United States).