A600669 0250

The whole cell lysate samples were run on BeyoGel™ Plus Precast PAGE Gel (4 to 10%) for Tris-Gly System (P0465S, Beyotime) for 110 min at 110 V. Gels were transferred to PVDF membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked by 5% (w/v) non-fat milk (A600669-0250, Sangon Biotech) at 4 °C overnight, before sliced into stripes according to a pre-stained ColorMixed Protein Marker (PR1920, Solarbio). The sliced stripes were incubated with primary antibody for 1 h at ambient temperature. β-actin were used to be a reference protein for Western blot in this project. Then the membranes were washed five times with TBST buffer before incubation with secondary antibody for 30 min at ambient temperature. The membranes were washed three times and imaged via ImageQuant LAS4000 (Cytiva). For semi-quantitative analysis, band intensities were measured by densitometry using the software ImageJ 1.38X. Primary antibodies used in this study include: FLAG tag mouse monoclonal antibody (1:20,000) (66008-3-Ig, Proteintech) and β-actin mouse monoclonal antibody (1:5000) (66009-1-Ig, Proteintech). Secondary antibody used in this study is peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (1:10,000) (SA00001-1, Proteintech).

Bacterial samples were resuspended in 1× protein loading buffer and boiled at 98 °C for 8 min. 0.05 OD of proteins samples were loaded each lane. Proteins were transferred onto PVDF membranes (#10600023, Cytiva) for 45 min at 150 V in transfer buffer. Membranes were blocked for 1 h at room temperature in 1× TBST buffer with 5% (w/v) skim milk (#A600669-0250, Sangon), washed with TBST twice and incubated with monoclonal α-FLAG (Sigma-Aldrich #F1804-5MG; 1:10,000), α-SipC (1: 3,000) or α-GroEL (Sigma-Aldrich #G6532; 1:10,000) antibodies for 1 h at room temperature. After three TBST washes, membranes were incubated with secondary α-mouse or α-rabbit HRP-linked antibodies (Sangon #D110087 or #D110058; 1:10,000) for 1 h at room temperature. Chemiluminescence was developed using the high sensitive ECL luminescence reagent (#C500044-0100, Sangon), and then visualized on ChemiScope 6000SE and quantified using ImageJ Software.

After washing with PBS three times, cells were harvested. Collected cells were treated with RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 5 mM EDTA, 1 mM NaF) with phosphatase and protease inhibitor cocktail (B15002 and B14002, BIMAKE, Houston, TX, USA), and sonicated to release the contents. The protein concentrations were determined using Bestbio BCA protein assay kit (BB-3401-3, Shanghai, China). Total protein (10 µg) was separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). After incubation in 5% non-fat milk (A600669-0250, Sangon Biotech, Shanghai, China) solution for one hour, the membranes were incubated in the primary antibodies GAPDH (1:5000) and DHODH (1:8000) at 4 °C overnight. The proteins were visualized by using the ECL Reagent (170-5061, Bio-Rad, Hercules, CA, USA) on ChemiDoc Touch Imaging system (Bio-Rad, Hercules, CA, USA).