Bradford protein assay kit

The ready Prep^TM protein extraction kit (Bio-Rad Inc., Catalog No., 163-2086) was used to extract total protein according to the manufacturer’s instructions. Bradford Protein Assay Kit (Bio Basic Inc., Markham Ontario, Canada) was utilized to determine protein concentration following the manufacturer’s instructions. For blotting, 20 μg protein was mixed with an equal volume of 2× Laemmli sample buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125M Tris HCl, pH 6.8), where the mixture was boiled at 95 °C for 5 min before loading on polyacrylamide gel. The blot was run, followed by membrane blocking at room temperature for 1 h. Primary antibodies of total and phosphorylated Akt were diluted in TBST and incubated overnight with the membranes at 4 °C. The blot was rinsed 3–5 times for 5 min with TBST and then incubated with the HRP-conjugated secondary antibody (Goat anti-rabbit IgG- HRP-1mg Goat mab-Novus Biologicals) for 1 h at room temperature. After another wash with TBST, the chemiluminescent substrate (Clarity TM Western ECL substrate Bio-Rad cat#170-5060) was applied and the signals were captured using a CCD camera-based imager. Image analysis software (Image J) was used to read total-Akt and P-Akt band intensities.

Hepatic tissues were homogenized in RIPA lysis buffer, and quantitative protein analysis was determined by Bradford protein assay kit (Bio Basic Inc., Ontario, Canada). Twenty μg of extracted protein from the liver of all studied groups was separated by SDS-PAGE on 4–20% polyacrylamide gradient gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes using Bio-Rad Trans-Blot Turbo. After incubation in 5% nonfat dry milk, Tris-HCl, 0.1% Tween 20 for 1 hr, primary antibodies were added and incubated at 4°C overnight. The primary antibodies used were TLR4, HMGB1, NF-κB, and caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Appropriate secondary antibodies were incubated for 2 hr at room temperature. After being washed twice in 1 × TBS-T, densitometric analysis of the immunoblots was performed in all studied samples against the control sample of housekeeping protein beta-actin by protein normalization on the ChemiDoc MP imaging system.

Collagen content in the lung was evaluated by Clarity™ Western ECL substrate provided by Bio-Rad Laboratories, Inc., Canada.
The ReadyPrep™ protein extraction kit (total protein) provided by Bio-Rad Inc. was employed and added to lung samples. (Catalog #163–2086) and Bradford Protein Assay Kit (SK3041) for quantitative protein analysis was provided by Bio Basic Inc. (Markham, Ontario, L3R 8T4 Canada). A Bradford assay was performed according to the manufacturer's guidelines to evaluate every sample's protein content. After that, an equal volume of 2 × Laemmli sample buffer containing 4% SDS, 10% 2-mercaptoehtanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris HCl was added to each sample's 20 g protein concentration. The pH was measured and adjusted to 6.8. Before loading on polyacrylamide gel electrophoresis, each prior mixture was heated at 95 °C for 5 min to achieve protein denaturation.

The frozen liver and kidney samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer supplemented with proteinase inhibitors. The homogenate was centrifuged at 10000 rpm, and the supernatant was collected, and protein concentration was determined using the Bradford protein assay kit (BioBasic, Markham, Canada). Forty µg of protein from each sample was electrophoresed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline/Tween 20 (TBST) for 1 h at room temperature (RT). The blocked membranes were probed with primary antibodies against TLR4, MyD88, NF-κB p65, p-JAK2, JAK2, p-STAT3, STAT3, suppressor of cytokine signaling 3 (SOCS3), cleaved caspase-3, PPARγ, Nrf2, HO-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C. After washing with TBST, the membranes were incubated with secondary antibodies and developed by an enhanced chemiluminescence kit (BIO-RAD, Hercules, CA, USA). The bands were quantified by ImageJ (version 1.32j, NIH, USA) [28 (link)], normalized to GAPDH and presented as a percent of the control.

Skin biopsies were lysed in Radioimmune precipitation assay (RIPA) lysis buffer PL005 (Bio BASIC, Canada). Total protein was measured using Bradford Protein Assay Kit for quantitative protein analysis (BIO BASIC, Canada) according to the manufacturer's instructions. Samples were run on sodium dodecyl sulfate (SDS)-polyacrylamide gel using TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, USA), then transferred onto nitrocellulose membranes and incubated with TNFAIP3 antibodies (Thermo Fisher Scientific, USA). Blots were then incubated with peroxidase-conjugated secondary antibodies (Novus Biologicals, Canada).The chemiluminescent substrate (Clarity Western ECL substrate—BIO-RAD, USA) was applied to the blot according to the manufacturer’s recommendations. The chemiluminescent signals were captured using a charge-coupled device (CCD) camera-based imager (BIO-RAD, USA). Image analysis software was used to read the band intensity of the target proteins relative to a control sample by total protein normalization on the ChemiDoc MP imager.

RAW264.7 cells (1 × 10⁶ cells/well) were cultured in a 12-well plate, pretreated with various concentrations of PLH (0–50 µg/mL) for 12 h, and stimulated with RANKL (100 ng/mL) for 10 min. Whole cell extracts were prepared with RIPA lysis buffer. Nuclear fractions were prepared using the nuclear extraction buffer. Protein concentration was determined using the Bradford Protein Assay Kit (Bio Basic Inc., Markham, ON, Canada). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose membranes. After blocking with 5% BSA, the bands were probed with specific primary antibodies and incubated at 4 °C overnight. HRP-conjugated secondary antibodies were then added. After incubation, the targeted proteins were visualized by enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA, USA). Images were exposed to the X-ray film (GE Healthcare Ltd., Chicago, IL, USA). PARP and β-actin were used as the internal reference.