Image studio 2

Fifty micrograms of protein were separated on 4–20% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes. Membranes were then blocked for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline. Thereafter, the membranes were incubated at 4°C overnight with primary mouse anti-IgG antibody (1:2,000; Cat. No. 7076; Cell Signaling Technology), rabbit anti-ZO-1 antibody (1:500; Cat. No. 61-7300; Invitrogen), rabbit anti-occludin antibody (1:500; Cat. No. 61-7300; Invitrogen), rabbit anti-JAM-A antibody (1:5,000; Cat. No. ab52647; Abcam), goat anti-Collagen IV antibody (1:500; Cat. No. 1340-01; Southern Biotech), rabbit anti-MMP-9 antibody (1:1,000; Cat. No. sc-6841R; Santa Cruz Biotechnology), rabbit anti-MPO antibody (1:1,000; Cat. No. sc-16128-R; Santa Cruz Biotechnology), or mouse anti-β-actin antibody (1:10,000; Cat. No. A1978; Sigma). The membranes were then washed with TBST three times at 5 min intervals, incubated with goat anti-rabbit IRDye 800CW (1:30,000; Li-Cor, Lincoln, NE), donkey anti-mouse IRDye 680LT (1:40,000; Li-Cor), or donkey anti-goat IRDye 800CW (1:30,000; Li-Cor) secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized and densitometrically analyzed using Odyssey infrared scanner and Image Studio 2.0 software (Li-Cor).

Protein samples were dissolved in SDS sample buffer (Laemmli), heated to 96 °C for 5 min, and loaded onto a 4–15% Criterion TGX Stain-Free Precast gel (Bio-Rad). The gel-separated proteins were imaged with the Gel-Doc EZ system (Bio-Rad), directly transferred onto polyvinylidene difluoride membrane and probed with various antibodies (Supplementary Methods). Scans were acquired with the Odyssey Fc system (Li-Cor), and analysed using Image Studio 2.0 software (Li-Cor). Immunoblot band intensities were normalized to the total amount of protein loaded, as quantified using Image Lab 3.0 software (Bio-Rad).

For protein detection, samples were subjected to SDS PAGE using SERVAGel™ TG PRiME™ 8–16% precast gels, transferred onto a PVDF membrane (Bio-Rad), and probed with primary antibodies anti-Lep^{44 (link)} (1:10,000), anti-SipB^{40 (link)} (1:1000), anti-InvJ^{40 (link)} (1:5000), anti-YidC^{45 (link)} (1:10,000), anti-OmpA^{46 (link)} (1:20,000), anti-ProW_N^{47 (link)} (1:10,000), and M2 anti-FLAG (1:10,000). Secondary antibodies were goat anti-mouse IgG DyLight 800 conjugate (1:10,000) and goat anti-rabbit IgG DyLight 680 (1:10,000). Scanning of the PVDF membrane and image analysis was performed with a Li-Cor Odyssey system and image Studio 2.1.10 (Li-Cor).

For protein detection, samples were subjected to SDS PAGE using SERVAGel^™ TG PRiME^™ 8–16% precast gels, transferred onto a PVDF membrane (Bio-Rad), and probed with primary antibodies anti-HA (1:1,000). Secondary antibodies were goat anti-rabbit IgG DyLight 800 conjugate (1:10,000). Scanning of the PVDF membrane and image analysis was performed with a Li-Cor Odyssey scanner and Image Studio 2.1.10 (Li-Cor).

For protein detection, samples were subjected to SDS PAGE using SERVAGel TG PRiME 8–16% precast gels, transferred onto a PVDF membrane (Bio-Rad), and probed with primary antibodies anti-SipB, anti-InvJ, anti-PrgJ, anti-SpaP, anti-MBP, anti-RNApol, and M2 anti-FLAG. Secondary antibodies were goat anti-mouse IgG DyLight 800 conjugate and goat anti-rabbit IgG DyLight 680. EPEA-tagged SpaP was visualized using CaptureSelect-biotin anti C-Tag conjugate and Streptavidin DyLight 800. Scanning of the PVDF membrane and image analysis was performed with a Li-Cor Odyssey system and image Studio 2.1.10 (Li-Cor).

Eight million cells of each MEF cell line were collected before confluence (n: 5). Ice-cold radio-immunoprecipitation assay (RIPA) buffer was utilized to lyse samples for 15 min at 4 °C followed by centrifugation at 20,000 g at 4 °C for 20 min. Protein extracts (30 μg) were separated on 4–20% Tris-Glycine Gel and electron-transferred to nitrocellulose membranes according to standard procedures. After blocking the free binding sites with 5% BSA (bovine serum albumin) reconstituted in phosphate-buffered saline with 0.2% Tween-20, the membranes were probed with anti-Opa1 (ab42364) and anti-tubulin-α (EP1332Y) antibodies. Anti-mouse and anti-rabbit fluorescence (1:10,000) (ab186695 and ab186696, respectively) were used as secondary antibodies. The images were quantitatively acquired using Image Studio 2.1 software (Li-Cor, Lincoln, NE, USA).