Westernbright ecl kit

Radioimmunoprecipitation assay lysis buffer was used for cell lysis and total protein extraction. The total protein content was quantified with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The protein extracts were combined with protein loading buffer and boiled for 10 min. Then, 15 μg of each protein sample was separated on a 10% sodium dodecyl sulfate polyacrylamide gel (80 V for 40 min, 120 V for 80 min) (Beyotime) and transferred to a polyvinylidene difluoride membrane (210 mA constant current, 60 min) (Millipore). The membrane was blocked with 5% skimmed milk at room temperature for 2 h and then incubated with anti-CDK4 (1:1000, Cell Signaling Technology, USA, 12790s) or anti-GAPDH (1:500, Bioss, China, bs-0755R) primary antibodies overnight at 4° C. The membranes were subsequently incubated with goat anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase (1:5000, Bioss, bs-0295G-HRP) at room temperature for 2 h. Finally, the proteins were visualized with a Western Bright ECL Kit (Advansta, USA).

Mtb H37Rv cultures grown under different stress conditions were used to harvest both cells and supernatant. The cells were resuspended in PBS and lysed using a bead beater for 10-20 cycles of 1 min pulse and 2 min pause. The culture supernatant was passed through a 0.2 μm filter. The protein concentrations (of both cell lysates and culture filtrate) were quantified by Bradford assay. Subsequently, the samples were fractionated on SDS-PAGE and transferred onto a nitrocellulose membrane (PALL, BioTrace™ NT nitrocellulose, USA) and confirmed by ponceau (Fluka Analytical, USA) staining. The blots were blocked with 5% skimmed milk (HIMEDIA, India) for 1 h at room temperature, followed by 3 washes with PBS and then incubated with primary antibody overnight at 4°C. The next day, the blot was washed thrice with PBS and further incubated with appropriate secondary antibody for 2 h and developed using a WesternBright™ ECL kit (Advansta, K-12045-D20) in a Biorad ChemiDoc instrument. The secreted levels of EspR were quantified by densitometry using Image J and represented in terms of fold change normalized to the total protein loaded in each condition.

Cell lysates were evaluated for the expression of RIG-I and RNA polymerase III subunit A by immunoblot analyses. Blots were incubated with a rabbit polyclonal antibody against RIG-I (Abgent, cat# AP1900a), a rabbit monoclonal antibody against RNA polymerase III subunit A (Cell signaling, cat# 12825S, clone D5Y2D), a rabbit monoclonal antibody against GAPDH (Cell Signaling, cat# 5174S), a rabbit monoclonal antibody against histone 3 (H3; Cell Signaling, cat# 4499S), or a rabbit monoclonal antibody against COX IV (Cell Signaling, cat# 9367), overnight at 4°C. Blots were washed and incubated in the presence of a HRP-conjugated secondary anti-rabbit antibody. Bound antibody was detected with the WesternBright ECL kit (Advansta). Immunoblots were then reprobed with a mouse monoclonal antibody against β-actin (Abcam, cat# 49900) to assess total protein loading. Immunoblots are representative of at least three separate experiments.

Cells were lysed with RIPA (Radioimmunoprecipitation Assay) buffer (50mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) plus protease inhibitor cocktail (Roche, Mannheim Germany) and phosphatase inhibitors (phosphatase inhibitor cocktail 1 from Sigma-Aldrich). Lysates were loaded onto SDS-PAGE and separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane from Millipore (Billerica, MA, USA) and probed with the primary antibody diluted in 2% milk in PBS (Phosphate-Buffered Saline) followed by HRP-conjugated secondary antibody, as previously described [23 (link)]. Bands were visualized using Western blot Luminol Reagent (Santa Cruz), WesternBright ECL kit (Advansta, Menlo Park, CA, USA), Clarity (BioRad, Hercules, CA, USA), or Clarity Max (BioRad, Hercules, CA, USA), and signals were captured on a film or using Bio-Rad ChemiDoc MP Imaging Systems (Hercules, CA, USA).

5 × 10⁶ YTS-CLEC16A and YTS cells were lysed in ice cold IP-lysis buffer. After centrifugation at 13,000 rpm for 10 min at 4°C, supernatants were pre-cleared with 50 μl of agarose G beads (Invitrogen) and IgG for 45 min at 4°C. The pre-cleared lysates were incubated with CLEC16A, Vps-16A, CD226, Hrs, Actinin-4, Nrdp1, IgG (Millipore) antibodies for 1 h at 4°C and then with 50 μl of agarose G beads for an additional 1 h at 4°C. Immune complexes were washed three times, dissolved in SDS sample buffer, electrophoresed, and transferred onto nitrocellulose membranes (Invitrogen). Western Blot analysis was performed as described above. Membranes were probed for CLEC16A (Abgent), Vps-16A (Santa Cruz), CD226, Hrs, and Nrdp1 (Novus BioLogicals). For CLEC16A in murine splenic cells, spleens were harvested and cell suspensions were prepared by dicing spleens. 250 × 10⁶ splenocytes from control and knockout mice were lysed in ice cold IP-lysis buffer and IP pull down was performed as described above. Membranes were probed for CLEC16A and Hrs. The membranes were washed and incubated with a respective mouse/rabbit secondary antibody and bound antibody was detected with WesternBright ECL kit (Advansta). Membranes were stripped and re-probed for β-actin as a loading control.