Ab82592

For the proteinase K digestion assay, equal amounts of MS_WT and MS_Rv0790c were collected and incubated with 100 μg/mL proteinase K at 37°C. PMSF (100 nM) was added at different time points to stop the reaction. Equal volumes of these bacteria were harvested and analyzed by western blotting, using an anti-Flag antibody for Rv0790c. For the bacterial plasmolysis assay, MS_WT and MS_Rv0790c bacterial cultures were ultrasonicated, and the cell lysates were centrifuged at 3,000 × g for 5 min. The supernatant was ultra-centrifuged at 27,000 × g for 40 min, after which they were identified as membrane-cytosol fractions and the pellets were identified as cell wall fractions. The expression of Rv0790c protein in each fraction was detected by western blotting using an anti-Flag antibody. GroEL, a mycobacterial cytosolic marker, was detected using an anti-GroEL antibody (Abcam, ab82592).

Protein samples were boiled in 1 × SDS loading buffer and separated using a 4–12% gradient Bis-Tris Gel (M00653; GenScript) by SDS-PAGE. Subsequently, the separated proteins were transferred onto a 0.45 µm PVDF membrane (IPVH00010; Millipore) and blocked with 5% skimmed milk at room temperature for 2 h. Then, the blots were incubated for another 2 h at room temperature with the following primary antibodies: anti-FLAG (1:1000, F1804; Sigma), anti-GFP (1:1000, K200047M; Solarbio), anti-cytochrome c (1:1000, MA5–11674; Invitrogen), anti-Drp1 (1:1000, ab184247; Abcam), anti-SipA (Shanghai Willget Biotech Co., Ltd.), anti-GroEL (1:1000, ab82592; Abcam), anti-Tomm20 (1:1000, ab56783; Abcam), and anti-β-Actin (1:1000, CW0096; CWBIO). To detect proteins, the blots were incubated with the corresponding horseradish peroxidase (HRP)-conjugated anti-rabbit (EF0002; SparkJade) or anti-mouse (EF0001; SparkJade) secondary antibodies at room temperature for 1 h. Finally, the blots were visualized with an ECL detection kit (D601039; Sangon Biotech) using an Amersham^TM Imager 600 system (General Electric). Immunoblotting bands were quantified using the ImageJ software. The data were collected from three biological replicates.

Whole-cell lysates of bacterial cultures were prepared as described previously and analyzed by SDS-PAGE (12%) for immunoblotting. Mouse anti-serum against TlyA was used at a dilution of 1:10000, and monoclonal antibodies of anti-GroEL (BEI resources, NR-13813) and anti-HBHA (BEI resources, NR-13804) at 1:500 dilutions. The anti-mouse peroxidase-conjugated antibody (#7076 cell signaling) was used as a secondary antibody. The E. coli specific anti-GroEL (Abcam ab-82592) and anti-β-lactamase (Abcam ab-12251) antibodies were used at a dilution of 1:500.