Luminata forte substrate

To determine the relative amount of PLY released between parental and mutant strains, sterile-filtered bacterial supernatants from cultures grown to mid-log phase (OD₆₀₀ of 0.5) were denatured by boiling for 5 min and separated on 10 % SDS-PAGE gels (Bio-Rad) prior to being transferred onto a 0.22 nm PVDF membrane (Millipore). The membranes were then blocked for 30 min (5 % milk) and probed with rabbit polyclonal anti-PLY antibody overnight. The blot was subsequently washed with and probed with goat anti-rabbit HRP conjugated secondary antibody (BioRad) for 1 h. Blots were incubated with Luminata Forte substrate (Millipore) for 1 min at 25 °C. The membranes were then developed following exposure to radiography film (GeneMate). Band density was calculated using ImageJ software after scanning film at 600 dpi (NCBI).

After glucose, palmitate or cytokine treatment, as described for mouse islets above, Ins-1 protein extracts^{23 (link)} were separated by 10% SDS-PAGE (Bio-Rad) then transferred to a polyvinylidene difluoride (PVDF, Bio-Rad) membrane. For Western blotting (WB), membranes were blocked in PBS/Tween plus 5% nonfat dry milk for 1 hr, followed by incubation with α-LDB1 (sc-11198x, 1:1000; Santa Cruz Biotechnology, Inc), α-MAFA (1:1000, NBP1–00121, Novus), α-ISL1 (1:1000, 39.4D5-c; Developmental Studies Hybridoma Bank), or α-NKX6.1 (1:1000, F55A10-c; Developmental Studies Hybridoma Bank) antibodies overnight at 4°C. A rabbit β-Actin antibody was used for loading control (#4967S, Cell Signaling Technology). Western blot primary antibody information can be found in Supplemental Table 3. The PVDF membrane was washed and incubated with species-matched horseradish peroxidase-conjugated secondary antibodies (Promega or Santa Cruz Biotechnology) followed by addition of Luminata Forte substrate (Millipore) and visualized using Chemidox XRS + Imager (Bio-Rad). All WB experiments were performed with at least three different cell preparations.

Tissue lysates were prepared from flash-frozen two pooled rat PV and LA. Samples were homogenized in lysis buffer (in mM: 30 histidine, 250 sucrose, 1× protease inhibitor cocktail cOmplete ULTRA (Roche Diagnostics, Singapore), 0.6 PMSF and 1 DTT) on ice. Insoluble material was removed by centrifugation at 11,000× g for 15 min, and the protein concentration of the supernatants was quantified according to the Bradford protein assay.
Proteins (30 µg) were then separated by SDS-PAGE on 9% tris-glycine gels and transferred to PVDF membrane (0.45 µm; Millipore, Burlington, MA, USA) using a wet transfer unit. After blocking of the membrane with 7.5% BSA in 0.1% TBS-Tween for 1 h at room temperature, the proteins of interest were labeled by the primary antibodies anti-GIRK1 and anti-GIRK4 (rabbit polyclonals, Alomone Labs, Jerusalem, Israel) overnight at 4 °C. The membranes were then incubated 1 h at room temperature with anti-rabbit HRP conjugated (Jackson ImmunoResearch, Ely, Cambridgeshire, UK) secondary antibody. The immunoblots were developed with Luminata Forte substrate (Millipore) and G: Box Chemi XR5 chemiluminescence imaging system (Syngene, Bengaluru, India). The protein-signal densities were normalized to the corresponding β-actin-signal densities. The analysis of the western blotting images was performed using ImageJ software 1.53f.

Cells were lysed directly using Laemmli sample buffer, and whole cell lysates were denatured at 95°C for 10 minutes and separated on either Mini-Protean TGX 4–20% gels (Bio-Rad Laboratories Inc., Hercules, CA) or 10% polyacrylamide gels. Protein was transferred to nitrocellulose membrane (Bio-Rad), and blotted with primary antibodies to KRAS (Sigma-Aldrich, clone 4F3), phospho-ERK (Cell Signaling Technology, #4377), total ERK (Cell Signaling Technology, #9102), phospho-Akt (Cell Signaling Technology, #4058), Akt (Cell Signaling Technology, #9272), EGFP (Santa Cruz Biotechnology, #SC-8354), and GAPDH (Santa Cruz Biotechnology, #FL-335). Blots were developed using HRP conjugated anti-rabbit or anti-mouse secondary antibodies and Luminata Forte substrate (Millipore, Billerica, MA).

Randomly chosen lower lobe pulmonary parenchyma portions were snap frozen in liquid nitrogen and stored at −80 °C until used. For protein isolation, samples were homogenized using a radio-immunoprecipitation assay lysis buffer (150 mmol/L NaCl, 1.0 % IGEPAL, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mmol/L Tris, pH 8.0) supplemented with protease/phosphatase inhibitors. After quantification (Pierce BCA Protein Assay Kit, Pierce Biotechnology, Rockford, IL, USA), 50 µg of proteins were loaded in 15 % sodium dodecyl sulfate polyacrylamide gels. Proteins were transferred into a polyvinylidene fluoride membrane following electrophoresis and incubated with the primary specific antibody for p27 (BD Biosciences, Santa Fe, CA, USA) overnight in Tris Buffer Saline + 0.1 % Tween + 5 % Bovine Serum Albumin. Total ERK 1-2 (Cell Signaling, Danver, MA, USA) protein was used as loading control. Blots were developed by chemiluminescence after HRP-secondary antibody, 1-hour incubation using Luminata Forte substrate (EMD Millipore, Billerica, MA, USA). Intensity of the bands was analyzed using the ImageJ 6.0 software (NIH, Bethesda, MD, USA) and the ratio between p27 and Total ERK 1-2 protein was calculated in densitometry units.

Cells were detached from culture dishes and homogenized in RIPA buffer plus protease inhibitor cocktail. The lysates were centrifuged at 4°C for 20 minutes (14 000 g) and supernatants were transferred to new tubes. Thirty micrograms of protein were used for each sample. SDS‐PAGE was performed at 100 V for 90 minutes in Tris/glycine/SDS buffer. Proteins were transferred onto a polyvinylidene difluoride membrane (Bio‐Rad) using a transfer apparatus at 100 V for 1 hour at 4°C in Tris/glycine buffer. After transfer, the membrane was washed with Tris/NaCl/Tween buffer (TBST) and blocked overnight at 4°C with 5% non‐fat milk in TBST. Next, the membrane was washed with TBST and incubated overnight with primary antibody for: mitofusin 1 (MFN) (orb11040; Biorbyt, Cambridge, UK), PINK (orb331233; Biorbyt), caspase‐3 (437800; Life Technologies), beta‐actin (A5441; Sigma‐Aldrich) and mitochondrial fision factor ‐MFF (orb325479; Biorbyt) at a dilution of 1:500. After washing the membrane, solution of appropriate secondary antibody conjugated with HRP was applied. After 2 hours incubation, the membrane was washed again with TBST and incubated with Luminata Forte substrate (Merck, Darmstadt, Germany) and visualized using chemiluminescence method with ChemidocMP (Bio‐Rad).

Protein extraction was prepared according to the protocol described above. The supernatant was collected and a volume of cooled acetone (− 20 °C) was added to the sample an incubated for 1 h at − 20 °C, the extract was centrifuged at 10,000 rpm for 10 min at 4 °C. The pellet was resuspended in phosphate buffer (50 Mm, pH 8.0), proteins were standardized by Bradford and a volume of 15 µL was injected into the electrophoresis pocket. A broad range of protein molecular weight was used as a marker. Electrophoresis was performed for 2 h at 120 V. The product was transferred to a nitrocellulose membrane (Merck Millipore Ltda, Tullagreen, USA) using a 300 A for 1 h. The membrane was incubated for 1 h with 5% of fat free milk prepared in TTBS, the membrane was rinsed 3 times with TTBS and were incubated for 1 h at room temperature with the primary antibody anti-DHN 1:1000 prepared in 3% of fat free milk in TTBS (Agrisera, Sweden) o were incubated also for 1 h with the primary antibody anti-RbcL 1:20,000, after the incubation the membrane was rinsed for 15, 10, 5 min with TTBS and the membranes were incubated with the secondary antibody anti-Rabbit IgG HRP conjugated (Agrisera, Sweden) de 1:10,000, after antibody treatment, the membranes were incubated for 3 min with Luminata Forte substrate (Merck, Tullagreen, USA) and the chemiluminescence were detected with x-ray films (Fujifilm).