Horseradish peroxidase conjugated igg

Protein was collected from the kidney using a cell lysis reagent plus a protease inhibitor (Roche, Basel, Schweiz). SDS-PAGE separated proteins in total kidney lysates (40 ug), which were then analyzed by Western blot. The designated proteins on the blots were probed with mAbs against α-SMA (1:1000 dilution, Abcam, Trumpington, Cambridge, UK), fibronectin (1:1000 dilution, Bioworld Tech, Nanjing, China), collagen IV (1:1000 dilution, Novus Biologicals, Littleton, CO, USA), and actin (Cell Signaling, Danvers, MA, USA), followed by horseradish peroxidase–conjugated IgG as the secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and visualized by enhanced chemiluminescence (Merck, Darmstadt, Germany).
The quantification of the Western blot was carried out according to the NIH ImageJ software (1.53k; National Institutes of Health; Bethesda, MD, USA) standard analysis method. The rectangular box (box tool) was used to select the entire band to be quantified. The signal peaks in the rectangular box were quantified, and the peak area was calculated as the IOD value of each band.

Western blot analysis was performed on cells treated with different concentrations of suramin sodium (suramin) (Wako Pure Chemical Industries, Osaka, Japan) (0, 20, 50, and 100 μmol/L) for 24 hours in DMEM without FBS. The cells were homogenized in RIPA buffer (25 mmol/L Tris‐HCl, pH 7.6, 150 mmol/L NaCl, 1% NP‐40, 1% sodium deoxycholate, and 0.1% SDS) with protease inhibitor cocktail (p8340, Sigma). The protein concentration was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Samples (20 μg) were separated on 10% TGX FastCast™ acrylamide gel (Bio‐Rad) and transferred to a Amasham™ Hybond™ 0.2 μm polyvinylidene fluoride (PVDF) membrane (GE Healthcare). Blocking of the membrane was performed using PBS with 5% skim milk powder (Wako Pure Chemical Industries). The antibodies used in this study were specific to HuR (sc‐5261, 1:2000, Santa Cruz, Santa Cruz, CA, USA), cyclin A (611268, 1:2000), cyclin B1 (554178, 1:2000), Cox2 (610203, 1:2000, BD Biosciences, Cambridge, MA, USA), and β‐actin (A5441, 1:10 000, Sigma). The secondary antibody was horseradish peroxidase‐conjugated IgG (Jackson ImmunoResearch Laboratories, 1:5000‐10 000, West Grove, PA, USA). The protein bands were visualized with the Amersham™ ECL™ start or Prime Western Blotting Detection Reagent (GE Healthcare) and detected by LAS‐4000 mini (GE Healthcare).

Cells and renal cortical tissues were collected and prepared as cytosolic fraction, membrane fraction, or total protein using a commercial kit (BioVision, Milpitas, CA, USA) as described previously [26 (link)]. Protein samples were quantitated using a commercial assay kit (Bio-Rad, Hercules, CA, USA). Samples were then separated on SDS polyacrylamide gels under denaturing conditions and electrophoretically transferred to polyvinylidenedifluoride membrane (Amersham-Pharmacia Biotech, Little Chalfont, UK). After blocking in 5% skimmed milk, the membranes were incubated overnight at 4 °C with antibodies against TRPV1, ALOX12, PKCα, Nox2, Nox4, NLRP3, IL-1β, caspase-1, actin, or Na⁺, K⁺-ATPase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the membranes were incubated for 1 h at room temperature with a corresponding horseradish peroxidase-conjugated IgG (Jackson ImmunoResearch, West Grove, PA, USA). After washing the membranes, the bound antibody complex was detected using a commercial enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). The density of the bands of appropriate molecular masses was determined semi-quantitatively by densitometry using an image analytic system (Diagnostic Instruments, Sterling Heights, MI, USA). The level of each protein was expressed relative to the amount of actin or Na⁺, K⁺-ATPase.

H/R-injured ECs were harvested after co-cultured with EPCs and/or NPCs. Proteins were extracted with cell lysis buffer (Thermo scientific) supplemented with complete mini protease inhibitor tablet (Roche). Protein lysates were electrophoresed through SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5 % non-fat milk for 1 h and incubated with primary antibodies against Akt (1:1000; Cell Signaling), p-AKt (1:1000; Cell Signaling), VEGFR2 (1:1000; Cell Signaling), p-Flk1 (1:200; Santa Cruz), and $\beta$ -actin (1:4000; Sigma) at 4 °C for overnight. After washing, membranes were incubated with horseradish-peroxidase-conjugated IgG (Jackson Immuno Research Lab) for 1 h at RT. Blots were developed with enhanced chemiluminescence developing solutions and quantified under ImageJ software. For detecting the protein expressions of Akt and p-AKt in all groups, two sets of gels were done. One set of gels was used to compare the difference between normoxia and hypoxia groups, and the other set of gels was used to compare the differences among different treatment groups in the Hypoxia groups. All experiments were repeated at least six times. Similarly, Es was calculated as: Es = (E_{EPC + NPC} − E_EPC − E_NPC) / (E_EPC +E_NPC) x 100 %.

Proteins from EC-EXs and EPC-EXs were isolated with lysis buffer (Thermo Scientific, FL) containing protease inhibitor. Protein concentration assay was conducted using a Bradford assay kit (Bio-Rad Laboratories). The linear range of the assay for BSA is from 0.2 to 0.9 mg/mL. Plates were read at 595 nm using a spectrofluorometer (BioTek Instruments). For western blot analysis, the proteins were subjected to electrophoresis and transferred onto PVDF membranes. The membranes were blocked by incubating with 5% dry milk for 1 hr, followed by incubation with primary antibodies overnight at 4°C. The primary antibodies used were anti-CD63 (1 : 400; BD Biosciences), anti-CD105 (1 : 500; Santa Cruz), anti-CD34 (1 : 500, Santa Cruz), and β-actin (1 : 4000, Sigma). After being washed thoroughly, membranes were incubated with horseradish peroxidase-conjugated IgG (1 : 40000; Jackson ImmunoResearch Labs) for 1 hr at room temperature. Blots were then developed with enhanced chemiluminescence developing solutions.