Goat anti rabbit igg hrp

Total cellular protein was extracted using the cell lysis buffer (Beyotime), and concentrations were determined by BCA protein assay kit (Beyotime). Lysates were loaded in SDS-AGE gel and electrophoresed. Proteins were transferred from gel to nitrocellulose membrane using a trans-blot electrophoretic transfer kit (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in 5% skim milk in TBST buffer and incubated with primary antibodies anti-JMJD6 (1:3000; H-7, Santa Cruz Biotechnologies, Dallas, TX, USA), anti-histone H4R3me2a (1:3000; active motif), anti-HA tag (1:4000; HA.C5, Abcam), anti-α-tubulin (1:6000, DM1A, CST), anti-PDE1C (1:2000; Abcam) and anti-GAPDH (1:4000; 6C5, Abcam). After washing, the membranes were incubated with HRP goat anti-mouse IgG (Beyotime) or HRP goat anti-rabbit IgG (Beyotime). Membranes were then incubated in BeyoECL plus (Beyotime) and then imaged using Chemidoc imaging system (Bio-Rad Laboratories). Gray value was analyzed using Image J (Java 1.8.0).

The tissue samples were fixed in 4% paraformaldehyde, hydrated through ethanol solution, embedded in paraffin and incubated with 3% hydrogen peroxide. Afterwards, the tissues samples were incubated with the primary anti-PHF5A (1:100 dilution, Abcam, ab193115) anti-FOS (1: 1:500 dilution, Abcam, ab184938), and anti-Ki67 (1:200 dilution, Abcam, ab16667) overnight at 4 °C. After incubation with secondary antibody HRP goat anti-rabbit IgG (1:200 dilution, Beyotime, A0208) at room temperature for 1 h, DAB and hematoxylin was stained. Scoring the intensity of tissue staining based on the criteria provided in the reference [22 ]. A score greater than or equal to the median of immunohistochemistry indicated high expression of PHF5A, otherwise low expression.

Total proteins were purified using RIPA lysis buffer (Beyotime, Jiangsu, China) and the concentrations were detected by protein assay kit (Beyotime, Jiangsu, China). Protein lysates were subjected to 10% SDS-PAGE, transferred to PVDF membrane (Millipore), hybridized with corresponding primary antibody (Additional file 3: Table S3) overnight at 4 °C. The next day, the membranes were incubated with secondary antibody HRP goat anti-rabbit IgG (Beyotime, A0208) at room temperature for 2 h. The coloration of the membrane was performed with chemiluminescence ECL kit (Thermo Fisher Scientific) and protein signal was visualized by Odyssey Infrared scanning system (Li-Cor, Lincoln, NE, USA).

The protocol was approved by the Institutional Ethics Committee of the Henan Provincial People’s Hospital, and informed consent of all patients was obtained. The pathological specimens were collected from 150 patients with gastric cancer who underwent primary surgical resection. The patients with gastric cancer treated surgically only without co-morbidities were included in the study. Notably, normal tissues used in the TMA were collected from paracancerous tissues of gastric cancer patients. TMA sections contained tumor tissues (n = 150) and matched normal tissues (n = 150), which were constructed according to the methods in the literature [19 (link)]. Formalin-fixed paraffin-embedded samples were cut into 5-µm sections and deparaffinized and rehydrated. Following the manufacturer’s protocols, the sections were treated with diluted primary antibody against CCNI2 (1:50, Thermo Fisher Scientific, PA5-35081) at 4 °C overnight and secondary antibody HRP goat anti-rabbit IgG (1:200, Beyotime, A0208) at room temperature for 30 min. subsequently, sections were stained by DAB and hematoxylin at room temperature to detect the signal. The intensity of CCNI2 positive staining in TMA sections was scored as previously describe [20 (link)]. Cases showing hybrid scores of more than or equal to the median were considered as CCNI2 high expression.

Western blotting analysis was conducted as previously described [16 (link)]. The protein was extracted from the hippocampal regions of mice with a lysis buffer at 4 °C for 1 min and centrifuged at 13,200 rpm for 30 min. The protein concentration in the supernatant was measured by Bradford assay. Samples were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteins were transferred to the polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in tris buffered saline with Tween (TBST) for 2 h, followed by overnight incubation with primary antibodies against RARα (1:800, Cat No: A19551, Abclonal, Wuhan, China) and β-actin (1:5000, Cat No: AC026, Abclonal). The membranes were washed three times (15 min for each) with TBST and incubated with secondary antibodies HRP goat anti-rabbit IgG (Cat No: A0208, 1:3000, Beyotime, Shanghai, China) at room temperature for 45 min. Blots were visualized with enhanced chemiluminescence according to the manufacturer’s instructions (American Bioscience, Aylesbury, UK). Data were evaluated as optical density. Then, the expression of proteins was evaluated by Image J.

Experimental cells were prepared and total protein was extracted using PBS (phosphate buffer saline) and 1×lysis buffer while the concentration and quality of total protein was detected using BCA Protein Assay Kit (Cat. #23225, HyClone-Pierce). Equal amounts of total protein were separated by 10 mL SDS-PAGE (polyacrylamide gel) consisting of 30% PAGE, 1.5 mol/L Tris, 10% SDS, 10% APS and TEMED. Then, the separated protein was transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was subsequently blocked with TBST solution containing 5% skim milk for 1 h at room temperature. Primary antibodies of targeted proteins were incubated with the membrane for another 2 h at 25 °C. After washed by TBST solution for three times (10 min/time), the second antibody (HRP Goat Anti-Rabbit IgG, 1:3000, Cat. #A0208, Beyotime) was added to the membrane for 2 h incubation at room temperature. Color development of each membrane was then performed using ECL-PLUS/Kit (Cat. #RPN2232, Amersham). Lastly, the membranes were undergone X-ray developing using ImageJ software with GAPDH as the negative control for all bands. Antibodies applied in WB assay were listed in Table S2.

After 21 days osteogenic differentiation of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs, the total cellular protein was extracted using the cell lysis buffer (Beyotime), and concentrations were determined by Bradford protein assay kit (Bio-Rad). Proteins were loaded in SDS-AGE gel and electrophoresed at 80 V for 30 min and 140 V for 60 min. Then, proteins were transferred from gel to nitrocellulose membrane using a trans-blot electrophoretic transfer kit (Bio-Rad). Membranes were blocked in 5% skim milk in TBST buffer for 60 min and incubated with primary antibodies osterix (1:3000, ab94744, abcam 45kd), collagen I (1:3000, ab34710, abcam 125kd), osteopontin (1:2000, ab166709, abcam 65kd), osteocalcin (1:2000, ab93876, abcam 11kd), Tubulin(1:4000, ab4074, abcam 50kd), phosphate-Akt (1:2000, 193H12, Cell signaling technology), Total-Akt (1:2000, C67E7, Cell Signaling Technology), phosphate-Erk1/2 (Thr202/Tyr204, Cell Signaling Technology), total- ERK1/2 (1:2000, Cell Signaling Technology). After washing with TBST buffer, the membranes were incubated with HRP goat anti-mouse IgG (1:3000, Beyotime) or HRP goat anti-rabbit IgG (1:3000, Beyotime). Membranes were then incubated with pierce ECL western blotting substrate (Thermo fisher) and then imaged using chemidoc imaging system (Bio-Rad).