Anti p iκb

SCs treated by heat stress, quercetin, or kaempferol were harvested and immediately lysed with RIPA lysis buffer containing a proteinase inhibitor cocktail. The total proteins were collected, and the concentrations were determined by a BCA protein assay. Equal amounts of protein were loaded and separated by 10% SDS-PAGE gels, and the proteins were then transferred onto Millipore polyvinylidene fluoride (PVDF) membranes and blocked with Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% skim milk for 2 h. The membranes were then incubated with primary antibodies, including anti-HSP70, anti-p-IκB, anti-IκB, anti-p-NFκB-p65, anti-NFκB-p65 (Cell Signaling, USA), anti-β-actin, anti-vimentin, and anti-occludin (Proteintech, Wuhan, China) antibodies, at 4 °C overnight. IκB, NFκB-p65, and β-actin expression levels were used as the controls. Antibodies were detected using HRP-conjugated secondary antibodies (Thermo Scientific, Waltham, MA, USA).

Cell lysates were prepared from 20 to 40 μg kidney tissue and cytoplasmic protein was obtained by centrifugation at 4°C. Equal aliquots of proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Primary antibodies were added and the membranes incubated at 37°C for 2 h with gently shaking. The primary antibodies included anti-TLR2 and anti-TLR4, both from Abcam; and anti-MyD88, anti-NF-κB, anti-p-NF-κB, anti-IκB, anti-p-IκB, anti-cleaved-caspase-3, anti-caspase-9, anti-Bax, and anti-Bcl-2, all from Cell Signaling Technology. After thorough washing, the membranes were incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch). Immunoreactive bands were visualized using an ECL system (Amersham Pharmacia). To control for lane loading, the same membranes were also incubated with anti-β-actin antibody (Epitomics), depending on the molecular weight of target proteins. The signals were quantified by scanning densitometry using a Bio-Image Analysis System (Bio-Rad), with each band normalized relative to the actin band in the same sample. The results from each experimental group were expressed as relative integrated intensity compared with that of the control measured with the same batch.

Pancreatic tissue samples from the various experimental groups were homogenized on ice with RIPA buffer (150 mM NaCl, 1% NP40, 50 mM Tris pH 8.0, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with a cocktail of protease inhibitors (Roche); The homogenized tissue was then centrifuged and the cleared supernatant was subjected to immunoblotting applying anti-cathepsin L (1:1000, Santacruz biotechnology, sc-6498), anti-pSTAT3 (Santacruz biotechnology, Sc-8059), anti-STAT3 (1:1000, Santa Cruz biotechnology, Sc-7179), anti-pIκB (1:1000, Cell signaling Technology, #2859), anti-IκB (1:1000, Cell signaling Technology, #4812) or anti-actin (1:10000, Sigma Aldrich, A1978) antibodies, essentially as described^{58 (link), 69 (link), 70} .

The levels of the target proteins were determined by performing Western blotting as reported previously [49 (link),50 (link)]. Briefly, compound-treated cell lysates were heated with an SDS loading buffer for 10 min at 95 °C and separated by SDS-PAGE. The proteins were transferred and blotted on a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and incubated with primary antibodies obtained from Beyotime (anti-COX-2, anti-iNOS, anti-p-iκB, anti-iκB, anti-p65, anti-p50, or anti-GAPDH) or obtained from Cell Signaling Technology (anti-p-p65, Beverly, MA, USA) and an HRP-conjugated secondary antibody purchased from Beyotime. The proteins were visualized by chemiluminescence according to the Beyotime ECL kit’s protocol and analyzed using ImageJ 1.53k software.

Renal tissue homogenates were prepared, and the supernatant was maintained at 4°C. Besides, 30 μg protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene fluoride (PVDF) membranes. The primary antibodies, including anti-cleaved caspase-3 (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-NF-κB (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-NF-κB (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-IκB (Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-p-IκB (Cell Signaling Technology, Inc., Danvers, MA, USA) were added and incubated at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Immunoreactive bands were visualized using ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). For the loading control, the same membranes were simultaneously probed with anti-GAPDH (Abcam, Cambridge, UK). The signals were quantified by scanning densitometry using a Bio-Image Analysis System. Relative protein expression was subsequently quantitated by normalizing to GAPDH levels using the Image-Pro plus 6.0 software.