Hrp conjugated secondary antibody

Human Bax, Bcl-2 and cleaved caspase-3 antibodies were from Cell Signaling Technology (USA). Cyclin D1, Cyclin A, Cyclin B1, CDK1, phospho-p65, phospho-ERK, phospho-AKT, MMP-2, MMP-7, and MMP-9 antibodies were purchased from Proteintech (USA). GAPDH antibody and HRP-conjugated secondary antibodies were purchased from BIOSS (Beijing, China). Ophiorrhiza pumila was purchased from Foshan Renhui Pharmaceutical Technology Co. (Foshan City, Guangdong Province, China).

The expression plasmids pcDNA3.1-NFAT2 and its empty vector pcDNA3.1 were purchased from promega (WI, USA). The Lipofectamine 2000 was obtained from Invitrogen (Thermofisher Scientific., USA). Pluronic F-127 was bought from Genecopoeia (MA, USA). Fluo-4/AM, anhydrous dimethyl sulfoxide (DMSO) and cell counting kit-8 (CCK-8) were acquired from Dojindo Molecular Technologies Inc. (Tokyo, Japan). Antibodies against NFAT2 (RRID: AB_2152507), Egr2 (RRID:AB_1139730), FasL (RRID:AB_302235), ERK (RRID:AB_11157324) and β-actin (RRID:AB_764434) were obtained from Abcam (Cambridge, UK). Antibodies against AKT (RRID:AB_329827), p-AKT(Try326) (RRID:AB_1264114), p-ERK(S189) (RRID:AB_490903), c-myc (RRID:AB_2151827) and Cox-2 (RRID:AB_2084968) were bought from Cell Signaling Technology (MA, USA). The HRP-conjugated secondary antibody was acquired from Bioss, Inc. (Beijing, China). Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (NJ, USA). The Click-iT Plus 5-ethynyl-2′-deoxyuridine (EdU) Flow Cytometry Assay kit was bought from Invitrogen. All chemical reagents used in this study were of analytical grade and all reagents for cell culture were purchased from Gibco BRL Life Technologies (MD, USA).

The cells were lysed in RIPA buffer (Sparkjade, Jinan, China) supplemented with protease inhibitor cocktail (Servicebio, Wuhan, China) and phosphatase inhibitors A and B on ice for 30 min. The lysates were centrifuged for 15 min at 12,000 rpm, and their protein content was measured. Equal amounts of protein per sample were resolved on 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% BSA in TBST for 2 h at room temperature, the membranes were incubated overnight with antibodies targeting GAPDH (Abclonal, Wuhan, China, AC002), RRS1 (Abcam, Cambridge, UK, AB188161), AEG-1 (Abcam, Cambridge, UK, ab227981), ABCG2 (ZenBio, Research Triangle Park, NC, USA, R26465), MDR1 (Proteintech, Wuhan, China, 22336-1-AP), ERK (ZenBio, NC, USA, 340373), p-ERK (ZenBio, NC, USA, 340767), BAX (CST, Danvers, MA, USA, #89477), Bcl-2 (CST, Danvers, MA, USA, #3498), BAD (Abcam, Cambridge, UK, ab32445), p-BAD (Abcam, Cambridge, UK, ab129192) and ubiquitin (Proteintech, Wuhan, China, 10201-2-AP) at 4 °C. The membranes were washed thrice with TBST and then incubated with HRP-conjugated secondary antibody (1:1000; Bioss, Beijing, China, bs-0295G-HRP), followed by three more washes with TBST. The positive bands were visualized via ECL (MDBio, Taiwan, China).

The expression of integrin αvβ3 was evaluated by western blot and normalized to that of β-actin [34 (link)]. Hth7, 8505C, THJ16T, Cal-62, and Nthy-ori 3-1 cells were plated in 6-well plates and cultured in cell-based medium containing 10% FBS for 24 h. Cells at ~ 80% confluence were used for subsequent protein extraction. The different cells were washed with 1 × PBS 3 times and then lysed in RIPA buffer supplemented with PMSF for 15 min at 4 ℃. Proteins were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The immunoblots were blocked with 1 × PBS-5% fat-free dried milk for 1.5 h at room temperature and then incubated at 4 ℃ with anti-integrin αvβ3 polyclonal antibody (1:1000, Bioss, China) and anti-β-actin antibody (1:5000, Abcam, UK) for more than 16 h. After incubation with the HRP-conjugated secondary antibody (1:5000, Bioss, China) for 1 h, the PVDF membranes were visualized with an enhanced chemiluminescence system kit (Millipore, Bedford, MA, USA), and the grayscale of the strip was analyzed by ImageJ [28 , 34 (link), 35 (link)].

Wilms tumor type 1 (WT1), a transcription factor, encodes a zinc finger protein that is associated with the development of renal failure, and located in podocyte nuclei, and is identified as a highly expressed specific marker in mature podocytes [31 (link)]. The podocyte number was estimated by staining WT1 in renal samples with immunohistochemistry as described previously [32 (link)]. In brief, the 4 μm paraffin-embedded sections of the renal cortex were stained with rabbit polyclonal WT1 antibody (1 : 100; ab180840; Abcam Cambridge, MA) overnight at 4°C. Then, the sections were incubated with an HRP-conjugated secondary antibody (Bioss, Beijing, China) for 1 h at room temperature. Subsequently, the sections were incubated with streptavidin HRP (Bioss, Beijing, China). The number of WT1-positive cells in 30 randomly selected glomeruli was recorded, and the mean value per glomerulus was calculated. The results were displayed as cells/glomerulus.

MTS cell proliferation colorimetric assay kit was purchased from Abcam (Cambridge, UK). Osimertinib was bought from Selleck Chemicals (TX, USA). UPLC grade methanol, acetonitrile, and analytical grade formic acid were acquired from Merk (Darmstadt, Germany). Deionized water was purified by the Milli-Q system (Bedford, MA, USA). Urea, trichloroacetic acid, iodoacetamide, and tris(s-carboxyethyl)-phosphine (TCEP) were purchased from Sigma-Aldrich (St. Louis, USA). Trypsin was obtained from Promega (WI, USA). Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (NJ, USA). Antibodies against pyruvate kinase PKM (PKM), integrin beta 3 (ITGB3), phosphoenolpyruvate carboxykinase (PCK2), and β-actin were obtained from Abcam (Cambridge, UK). Antibodies against ATP synthase protein (ATP8), plasminogen activator inhibitor 1 (SERPINE1), and glycine amidinotransferase (GATM) were bought from Cell Signaling Technology (MA, USA). The HRP-conjugated secondary antibody was acquired from Bioss, Inc. (Beijing, China). All reagents for cell cultures were bought from Gibco BRL Life Technologies (MD, USA) and all chemicals were analytical grade.

Cells were immediately washed with PBS and lysed in RIPA buffer with complete protease inhibitor (Sigma-Aldrich) immediately. Protein lysates in protein loading buffer were separated in 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, Germany), blocked with 5% milk, and then probed overnight at 4°C with primary antibodies, including anti-NLRC4 (ab115537; Abcam, UK) and anti-GAPDH (Bioss antibodies, Woburn, MA). After incubation with an HRP-conjugated secondary antibody (Bioss, USA), the blots were washed with TBST and imaged using an enhanced chemiluminescence system (Thermo Scientific, USA).