Mini trans blot cell system

Proteins were separated on precast Mini-PROTEAN TGX gradient gels (4–15% or 4–20%, Bio-Rad) or self-made Tris-glycine gels and transferred (200 mA for 90 min, or 55 V for 75 min) onto 0.45-μm Immobilon-IP polyvinylidene fluoride membranes (Millipore) or 0.45-μm nitrocellulose membranes (Millipore) using the Mini Trans-Blot Cell System (Bio-Rad). Protein Marker VI (10–245 kDa) prestained (AppliChem) was used. Membranes were blocked with 2.5% BSA in TBST (50 mM Tris HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20). Blots were incubated overnight at 4 °C with primary antibodies. Blots were washed three times (each 5 min) with TBST and incubated for 1 h at RT with secondary antibodies (HRP-conjugated for chemiluminescence). Subsequently, blots were washed twice with TBST and once with TBS (50 mM Tris HCl (pH 7.6) and 150 mM NaCl). For chemiluminescence visualization, blots were incubated with the ECL Prime Western blotting detection reagent (GE Healthcare) and detected with ChemiDoc Imaging System (Bio-Rad).

Heads from 2–3 d old flies were homogenized as described in [31] (link) and proteins separated on 8% or 12% SDS-PAGE gels and transferred using the Bio-Rad Mini Trans-Blot® Cell system. Proteins were transferred to Hybond membranes (Amersham Bioscienses). The antibody against SWS [24] (link) was used 1∶1000 and anti-PKA-C3 (kindly provided by B. Biteau, University of Rochester, New York) 1∶4000. All antibodies were diluted in TBST supplemented with 3% BSA. Bands were visualized using horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) at 1∶1000 and the SuperSignal® West Pico chemiluminiscent substrate (ThermoScientific). Homogenates from 10 fly heads were used per lane.

Whole cell lysates from bladder urothelium and cultured cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing proteinase inhibitors, which were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) using a Mini Trans-Blot Cell system (Bio-Rad Laboratories). Membranes were blocked with 5% bovine serum albumin diluted in TBST (BSA/TBST) and incubated with primary antibodies diluted in 1% BSA/TBST followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted in 1% BSA/TBST and developed for reading by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo). Images were acquired with the LAS-4000 imaging system (Fujifilm Life Science, Tokyo, Japan). Anti-Cx43 (C6219, Sigma-Aldrich, 1:8000), anti-BMAL1 (ab93806, Abcam, 1:500), anti-beta actin (ab6276, Abcam, 1:5000) and anti-GAPDH (GAPDH, 2118, Cell Signaling Technology, Danvers, MA, USA, 1:5000) were used as the primary antibodies.

After separation, proteins were transferred to a nitrocellulose membrane by western blotting (Mini Trans-Blot^® Cell system from Bio-Rad). The membrane was incubated for 1 h in blocking solution [137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄, 0.05% Tween, and 5% (w/v) skimmed milk powder]. After two washing steps with PBST (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄, and 0.05% Tween) for 5 min the nitrocellulose membrane was incubated for 1 h with antibodies against V5-MrpG, or Strep-NqrA, which were diluted 1:5000 in blocking solution. In both cases HRP-conjugated antibodies were used (V5 Tag Monoclonal Antibody E10/V4RR HRP, Thermo Fisher Scientific; Strep-Tactin^® conjugated to horseradish peroxidase, iba). After two washing steps with PBST, bound antibodies were detected using chemiluminescence Clarity^TM Western ECl Blotting Substrates (Bio-Rad). Exposure (2 min) and detection were performed with the Image Quant LAS 400 (GE Healthcare). For a chromogenic immunodetection, the membrane was incubated with 20 ml PBST, 200 μl 3% 4-Chloro-1-naphthol, and 20 μl 30% H₂O₂. As positive control for the immunostaining of Strep-NqrA, the holo NQR-ST complex expressed in V. cholerae from the pNqrST plasmid was used. The Strep-His-tagged NQR complex was purified by Nickel affinity chromatography as described for His-tagged NQR complex (Tao et al., 2008 (link)).

Equal amounts of proteins were run on 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) and blotted onto nitrocellulose membrane using a Mini Trans-Blot Cell system (Bio-Rad). Membranes were blocked for 1 h at room temperature (RT) with a filtered solution of 4% skimmed milk in 1xTBS containing 1% Tween20. After blocking, membranes were rinsed with TBS-T and incubated overnight at 4 ºC with primary antibodies (α-mt-Co1 Abcam #14705, α-Tom20 Abcam #186735, α-Vinculin Abcam #129002, α-ATP5A Abcam #14748, Sdha Abcam #14715, α-Pitrm1 Linaris #PAK0154). After washing in TBS-T, secondary HRP-coupled antibodies were incubated for 1 h at RT (1:5000 goat anti-rabbit and 1:2000 goat anti-mouse antibodies). Signal detection was performed with Clarity Western ECL Substrate (Bio-Rad) following the manufacturer’s instructions on a Gel Doc XR + Gel Documentation System (Bio-Rad).

Single retinas were homogenized and sonicated in lysis buffer and centrifuged. Sample buffer was added to the supernatant just prior to use. Known amounts of protein (10 to 20 μg) or protein ladder were loaded into each well of an SDS-polyacrylamide gel. The Bio-Rad mini-trans blot cell system and mini protean pre-cast gells at 4–20% were used (Hercules, CA). Loading controls included GAPDH (1:1000; ab9485, Abcam, Cambridge, MA) or HSP60 (1:1000; ab45134, Abcam). The protein was transferred onto nitrocellulose using the Bio-Rad trans blot turbo transfer system (Hercules, CA) and alkaline phosphatase was used for band detection. Band density was quantified by scanning the blot using an EPSON scanner and Adobe Photoshop to convert to grayscale and invert the image. Each band was selected with the same frame and set measurements were used to obtain the greay mean value for each. Antibodies used were anti-caspase 1 (1:1000; ab108362, Abcam), anti-SOD2 (1:1000; ab13533; Abcam), anti-TXNIP (1:1000; 14715, Cell Signaling Technology, Danvers, MA), anti-NLRP1 (1:1000; 4990, Cell Signaling Technology), and anti-NLRP3 (1:1000; 15101, Cell Signaling Technology).