Rabbit anti klf4

Crude proteins were extracted from VSMCs as described previously [32 (link)], resolved by SDS/PAGE, and transferred onto a PVDF membrane (Millipore). Membranes were blocked with 5% (w/v), nonfat, dried skimmed milk powder in TTBS (100 Mm Tris/HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20) for 2 h at 37°C and then incubated overnight at 4°C with the following primary antibodies: 1:300 dilution rabbit anti-apelin (GeneTex), 1:500 dilution rabbit anti-KLF4 (Abcam), and anti-β-actin and rabbit anti-IgG (Santa Cruz Biotechnology) antibodies. After incubation with the appropriate secondary antibody, the immunoreactive signals of antibody–antigens were visualized using the Chemiluminescence Plus Western Blotanalysis kit (Tanon Biotechnology).

Cells were lysed with RIPA buffer (Pierce, Rockford, IL, USA) The BCA Protein Assay Reagent (Pierce) was used to quantify the protein concentration of lysates. Equal amounts of protein were resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the proteins were transferred to the polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking for 4 h at room temperature in 10% nonfat dry milk in TBST containing 0.05% Tween-20, the membranes were incubated with primary antibodies overnight at 4 °C and then after washes using TBST, it was incubated with HRP-conjugated secondary antibodies for 2 h at room temperature.
Primary antibodies included rabbit anti-Klf4 (Abcam, Cambridge, UK, 1:1000) Rabbit monoclonal anti-GAPDH (Sigma, 1:5000) rabbit anti-Nanog (CST, Danvers, MA, USA), Anti-ERK1/2 rabbit polyclonal antibody (sangon biotech, Shanghai, China, 1:1000), Anti-ERK1/2(Thr202/Tyr204)) rabbit polyclonal antibody (sangon biotech, 1:1000), Rabbit Polyclonal anti-ABHD2 (Proteintech, Rosemont, IL, USA, 1:1000) and anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibody were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China ).

HCFs were cultured on 4-well cell culture slides (30,104; SPL Life Sciences, Pocheon, Korea). After fixation in 4% paraformaldehyde, the cells were incubated with 0.1% Triton X-100 in PBS for 30 min, blocked for 1 h with 1% bovine serum albumin in PBS, and incubated with rabbit anti-KLF4 (Abcam, Cambridge, UK) and rabbit anti- α-SMA (Sigma Chemical Co, MO, USA) antibodies. The secondary antibodies were anti-rabbit IgG and anti-mouse IgG (BioLegend, CA, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Chemical Co). Fluorescence images were obtained using an Olympus CKX41 microscope (Tokyo, Japan).

Total protein of J1 mESCs or F9 EC cells was extracted, and its concentration was determined using the bicinchoninic acid protein assay (Beyotime). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the proteins were transferred to the polyvinylidene fluoride membranes (Millipore). After blocking for 3 h at room temperature in 10% nonfat dry milk in TBST containing 0.05% Tween-20, the membranes were incubated with primary antibodies overnight at 4°C and then after washes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Peroxidase activity was detected through autography using SuperSignal west pico substrate (Pierce/Thermo Scientific, Rockford, IL, USA). Primary antibodies included rabbit anti-Klf4 (Abcam, Cambridge, UK, 1:1000) and anti-β-catenin (Santa Cruz, CA, USA, 1:1000). Rabbit monoclonal anti-PCNA (Abcam. 1:1000). Rabbit monoclonal anti-GAPDH (Sigma. 1:5000).

After the cells were fixed, permeabilized and blocked, they were incubated overnight at 4°C with primary antibodies, including rabbit anti-Lumican (1:500, Abcam, United Kingdom), rabbit anti-Fibronectin (1:200, Abcam, United Kingdom), rabbit anti-α-SMA (1:500, Abcam, United Kingdom), rabbit anti-Nanog (1:100, Abcam, United Kingdom), rabbit anti-Sox2 (1:250, Abcam, United Kingdom), mouse anti-Oct4 (1:100, CST, United States), rabbit anti-Klf4 (1:100, Abcam, United Kingdom), mouse anti-ABCG2 (1:50, Abcam, United Kingdom), rabbit anti-Pax6 (1:50, Abcam, United Kingdom), rabbit anti-Myosin Ⅱa (1:50, CST, United States), rabbit anti-HIF1α (1:800, CST, United States) and rabbit anti-YAP1 (1:200, Bioss, China) followed by washing three times in PBS and incubation with FITC-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (1:500, Life, United States) for 1 h at room temperature. Cells were then incubated with DAPI for nuclear staining for 15 min. Finally, imaging was performed using an LSM800 confocal microscope (Zeiss, Germany). The fluorescence intensity was quantified using ImageJ software (version 1.47, National Institutes of Health, United States).

LNCaP cells were starved overnight, then transfected with DsiRNAs for 48 h. Cells were fixed in ice-cold methanol for 4 min at –20 °C after 4 h of stimulation with DHT (10 nM). This was followed by three washes with PBS and incubation with the following antibodies diluted in blocking buffer (0.2% BSA (Bioshop), 0.1% Triton X100 (Sigma-Aldrich)): rabbit anti-FLAG (Sigma Aldrich, #F7425), mouse anti-AR (Santa Cruz, #7305), or rabbit anti-KLF4 (Abcam, #215036) for 1 h at room temperature. After washes in PBS, coverslips were incubated with Alexa 568-conjugated goat anti-rabbit (Thermo Fisher Scientific, #A11011) or Alexa 488-conjugated goat anti-mouse (Cell Signalling Technology, #4408) antibodies for 1 h at room temperature. They were washed twice with PBS before being mounted on slides using ProLong Gold antifade with DAPI (Thermo Fisher). Pictures were acquired with an Olympus FV1000 using the FluoView 3.0 software or with a Nikon Eclipse E600 imaging system using MetaView.