Peroxidase affinipure goat anti rabbit igg h l

Blood was collected by cardiac puncture from pFN KO and control (Fn(fl/fl)) mice after they were humanely killed. Plasma preparation was performed using EDTA-coated tubes (Sarstedt). Three microliter aliquots of plasma were analyzed by standard western blotting under reducing conditions. The polyclonal rabbit anti-mouse FN antiserum (Millipore #AB2033) was used 1:500 diluted as primary antibody, with the 1:200 diluted Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson Laboratories) as secondary antibody.

Cells were lysed in 1x RIPA buffer (Cell Signaling Technology) supplemented with 1× Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Protein concentration was determined via BCA protein assay (Pierce). Equivalent protein from each sample was separated by SDS–PAGE and transferred to polyvinylidene difluoride membrane by wet transfer at 4°C. Blots were washed in 1× TBS with 0.05% Tween-20 (TBST) and blocked for 1 h with 5% milk in TBST at room temperature. The blots were incubated overnight at 4°C with primary antibody (1:1,000) in 5% BSA in TBST, washed, and incubated with secondary antibody (1:5,000) with 5% milk in TBST for 1 h, washed, and developed with ECL Clarity Max (Bio-Rad Laboratories). Primary antibodies used in this study include: β-Actin (13E5) Rabbit mAb #4970, PTEN (138G6) Rabbit mAb #9559, Stat3 (79D7) Rabbit mAb #4904, Phospho-Stat3 (Tyr705) (D3A7) XP Rabbit mAb #9145, ERK1/2 Rabbit mAb #9102S, pERK1/2 Rabbit mAb #4370S, AKT Rabbit mAb #9272S, pAKT Rabbit mAb #9271S, vinculin Rabbit mAb #05386 (EMD Millipore). Secondary antibody is Peroxidase AffiniPure Goat Anti-Rabbit IgG (H&L) (111-035-003; Jackson Immunoresearch). Densitometries were determined using ImageJ.

Recombinant Human ACE2 Protein (933-ZN) was purchased from R&D Systems (Minneapolis, MN, USA) and stored in a buffer according to the manufacturer’s instructions. Monoclonal mouse anti-human ACE2 Antibody (MAB933) was purchased from R&D Systems (Minneapolis, MN, USA). Human recombinant VE-cadherin extracellular domain (EC1-5) was produced in the laboratory. Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (AB-2338447), Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (AB-2307391), and Cy3™ AffiniPure Goat Anti-Mouse IgG (H + L) (AB-2338680) were all purchased from Jackson Immunoresearch (Ely, Cambridgeshire, UK). Hoechst solution (33,258) was purchased from Sigma Aldrich. The mouse monoclonal anti-human VE-cadherin antibody (clone BV9) and the rabbit polyclonal anti-EC1 anti-human VE-cadherin antibody were produced in the laboratory.

Primary antibodies: rabbit anti-AP4 (TFAP4) serum (1:2000, a gift from Takeshi Egawa [5 (link)]); rabbit anti-LRP6 (C5C7) (1:500, Cell Signaling Technologies Cat. # 2560); mouse anti-ACTIN (clone C4) (1:500, EMD Millipore Cat. # MAB1501).
Secondary antibodies: peroxidase AffiniPure goat anti-rabbit IgG (H+L) (1:7500, Jackson ImmunoResearch Laboratories Cat. # 111-035-003); IRDye 800CW donkey anti-rabbit IgG (H+L) (1:10,000, Li-Cor Cat. # 925–32213); IRDye 800CW donkey anti-mouse IgG (H+L) (1:10,000, Li-Cor Cat. # 926–32212).
Primary and secondary antibodies used for detection with the Li-Cor Odyssey imaging system were diluted in a 1 to 1 mixture of Odyssey Blocking Buffer (Li-Cor Cat. # 927–40000) and TBST (Tris buffered saline (TBS) + 0.1% Tween-20), and those used for detection by chemiluminescence were diluted in TBST + 5% skim milk. Primary antibody incubations were done overnight at 4°C, and secondary antibody incubations were done for 1 hr at room temperature (RT).

Cells were collected by trypsinization and washed 3 times with PBS (1,000 × g, 5 minutes, 4°C). Cell pellets were resuspended in lysis buffer (1% (w/v) digitonin, 1× Roche cOmplete protease inhibitor, 0.5 mM PMSF, 10 mM Tris–HCL (pH 7.4)) and incubated on ice for 40 minutes. The lysates were then centrifuged (17,000 × g, 10 minutes, 4°C) and the post-nuclear fractions were transferred to new tubes. The protein concentration of each sample was determined by Bradford assay. Samples were adjusted with TBS buffer and 6× Laemmli buffer + 100 mM dithiothreitol (DTT) and heated at 50°C for 10 minutes. Samples were separated by SDS-PAGE and transferred to PVDF membranes (Merck), then blocked in 5% milk + PBS-T (PBS + 0.2% (v/v) Tween-20) for 1 hour. Blocked membranes were incubated with primary antibody (LRRC15 [LSBio aa393-422], ACE2 [Abcam Ab108252], GAPDH [GeneTex GTX627408]) in 5% milk + PBST (PBS + 0.2% (v/v) Tween-20) at 4°C overnight, then incubated with peroxidase (HRP)-conjugated secondary antibodies (Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) [Jackson ImmunoResearch 111-035-144], Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) [Jackson ImmunoResearch 115-035-146]) for 90 minutes at room temperature.