Western blotting (WB) was estimated according to a previous article [27 (link)]. The antibodies used in this study are listed as follows: anti-eIF2γ (1 : 500; PA5-31177, Thermo Fisher Scientific, MA, USA), anti-PCNA (1 : 500; BM0104, Boster, Wuhan, China), anti-Cyclin D (1 : 500; WL01435a, Wanlei, Shenyang, China), anti-GAPDH (1 : 2000; AC002, ABclonal, Wuhan, China), anti-ZBTB16 (1 : 500; No. D222893, BBI Life, Shanghai, China), anti-pERK (1 : 2000; #4370, CST, Boston, USA), and anti-ERK (1 : 2000; #9194, CST, Boston, USA). The results were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified using ImageJ (V1.48d).
Wl01435a
Manufactured by Wanlei
Sourced in China
The WL01435a is a laboratory equipment product manufactured by Wanlei. It is designed for general laboratory use. The core function of the WL01435a is to perform basic laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Ab124800, by Abcam (2 mentions)
Ab16148, by Abcam (2 mentions)
Wl02785, by Wanlei (2 mentions)
Wl02028, by Wanlei (2 mentions)
Wl03069, by Wanlei (2 mentions)
Km9001t, by Sungene Biotech (2 mentions)
Goat anti rabbit igg h l hrp, by Boster Bio (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (2 mentions)
Anti cyclin d, by Wanlei (1 mentions)
Pa5 31177, by Thermo Fisher Scientific (1 mentions)
Anti pcna, by Boster Bio (1 mentions)
Bm0104, by Boster Bio (1 mentions)
Imaging system, by Bio-Rad (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
Ac002, by ABclonal (1 mentions)
Ab62739, by Abcam (1 mentions)
Wl02117, by Wanlei (1 mentions)
Sds page loading buffer, by Beyotime (1 mentions)
Trans blot sd cell and systems, by Bio-Rad (1 mentions)
7 protocols using wl01435a
1
Western Blotting for Protein Expression Analysis
2
Immunohistochemical Profiling of Oncogenic Markers
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Immunohistochemistry (IHC) staining assay was performed as previously described66 (link). A total of 48 cancer tissue samples were obtained; 30 from patients from the 967rd Hospital of People’s Liberation Army and 18 from Qiqihar Medical University. The samples were incubated with a primary antibody against ELF5 (1:400, ab13581, Abcam, Cambridge, UK), SIRT6 (1:600, ab62739, Abcam, Cambridge, UK) or Cyclin D1 (1:400, WL01435a, Wanleibio, Shenyang, China). IHC staining assay was conducted according to the manufacturer’s instruction (ZSGB-BIO, Beijing, China). The IHC score of ELF5, SIRT6 and Cyclin D1 were quantified from 0 to 12 according to the percentage of stained tumor cells and staining intensity. Low expression was given a score of 0–2, moderate expression was given a score of 3–7, and high expression was allocated a score of 8–12. All individuals who donated the tissues for this study gave their consent in written form. Histologic immunohistochemical images for Figs. 5 f and 7 i were obtained with an Olympus IX83 Microscope Camera (Tokyo, Japan). The slides were scanned at ×20 magnification (scale bar, 500 μm).
3
Western Blot Analysis of Cell Signaling Proteins
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The cells were lysed by cold RIPA buffer (Beyotime, Shanghai, China) for 30 min on ice, added to 5× SDS-PAGE loading buffer (Beyotime), and heated at 100 °C for 10 min. The lysates were then separated by 8–12% SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes by Trans-Blot SD Cell and Systems (Bio-Rad) for 45 min at 15 V. After blocked with 5% non-fat milk in TBS-T buffer (20 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20), PVDF membranes were incubated with the primary antibodies [34 (link)]. After incubation with a secondary antibody, the signals were measured using a chemiluminescent imaging system (Tanon, Shanghai, China). Beta-ACTIN was used as an endogenous loading control. Antibodies used in this study were: Cyclin D1 (WL01435a, Wanleibio), CASPASE3 (WL02117, Wanleibio), β-Catenin (51067-2-AP, Proteintech, Chicago, IL, USA) AXIN1(16541-1-AP, Proteintech), AXIN2(20540-1-AP, Proteintech) and β-Actin (AC028, ABclonal, Wuhan, China).
4
Immunoblot Analysis of Cell Cycle Regulators
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Total proteins were extracted using cell lysis buffer (P0013, Beyotime) and separated by SDS‐PAGE. Then, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against SHMT1 (1:1000, D3B3J, CST), cyclinB (1:1000, 4138S, CST), CDC20 (1:1000, 4823S, CST), β‐actin (1:1000, A00702, GenScript), HOXD8 (1:1000, sc‐515357, SantaCruz), p21 (1:1000, A1483, ABclonal), γ‐H2AX (1:500, CSB‐PA010097OA139phHU, CUSABIO), p‐CDK1 (1:1000, CSB‐PA000492, CUSABIO), p‐CHK1 (1:1000, CSB‐PA006769, CUSABIO), p‐CDC25 (1:1000, CSB‐PA080107, CUSABIO), cyclinE (1:1000, sc‐377100, SANTA), and cyclinD (1:1000, WL01435a, WanleiBio) overnight at 4°C with shaking. Then, the membranes were washed with Tris Buffered Saline with Tween20 (TBST) and incubated with secondary antibody conjugated with HRP (1:5000, GenScript) for 1 h at room temperature. The signals were developed by the ECL detection kit (Wanleibio).
5
Bovine Myoblast Protein Expression Analysis
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Total bovine primary myoblasts proteins were extracted from different treatment groups using RIPA lysis buffer containing 1mM PMSF (Solarbio; Beijing, China). The protein extract was then boiled for 10 min in a loading buffer of 5× SDS-PAGE. The protein was then separated by SDS-PAGE and transferred to a 0.2 μm polyvinylidene fluoride membranes (PVDF). Then incubated for 2 h at room temperature with 5% skim milk in Tris Buffered Saline, with Tween (TBST) to block the PVDF membrane. Subsequently, in 4 °C overnight incubation with the primary anti-MYOD specific antibodies (Dilution 1:1000; ab16148; Abcam, Cambridge, UK), anti-MYOG (1;1000; ab124800; Abcam), anti-MYH2 (Dilution 1:1000; WL02785; Wanleibio, Shenyang, China), anti-CDK2 (Dilution 1:1000; WL02028; Wanleibio), anti-PCNA (Dilution 1:500; WL03069; Wanleibio), anti-CyclinD1 (Dilution 1:1000; WL01435a; Wanleibio), anti-β-actin (1:1000; KM9001T, SungeneBiotech, Tianjin, China). After that, the PVDF membranes were washed with TBST buffer and then incubated at room temperature for 2 h with the secondary antibody. The secondary antibodies were: goat anti-mouse IgG HRP (M21001S, Abmart, Shanghai, China), goat anti-rabbit IgG (H + L)-HRP (BA1054, Boster, Wuhan, China). Finally, ECL luminous liquid (DiNing, Beijing, China) was used to detect the protein bands.
6
Western Blot Analysis of Protein Markers
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Total protein was extracted by RIPA buffer (Beyotime, China) containing 1% PMSF (Beyotime, China) and was quantified using a BCA Kit (Beyotime, China) according to the instructions of the manufacturer. 40 mg of protein was fractionated by SDS-PAGE, transferred onto 0.45 μm PVDF membrane (Millipore, USA). The membranes were blocked with defatted milk powder (Biosharp, China) at room temperature for one hour and incubated with the appropriate primary antibodies. The specific primary antibodies against GNPNAT1 (16282-1-AP, Proteintech, 1:1000), α-Tubulin (66031-1-Ig, Proteintech, 1:20000), c-Myc (67447-1-Ig, Proteintech, 1:5000), Cyclin D1 (WL01435a, Wanlei, 1:1000), Bcl-2 (BF9103, Affinity, 1:1000), Bax (2772, CST, 1:1000), E-cadherin (ET1607-75, Huabio, 1:1000), N-cadherin (ET1607-37, Huabio, 1:1000), Vimentin (BS1491, Bioworlde, 1:1000) were used and incubated at 4°C overnight. After being washed for three times in TBST, the membrane was incubated with corresponding HRP-conjugated secondary antibodies (BL001A and BL003A, Biosharp, 1:10000) at room temperature for one hour. Immunoblots were visualized by gel and blot imaging system (Bio-Rad, USA) with enhanced chemiluminescence assay (Millipore, USA).
7
Proteomic Analysis of Bovine Myoblasts
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Total bovine primary myoblasts proteins were extracted from different treatment groups using RIPA lysis buffer containing 1 mM PMSF (Solarbio; Beijing, China). The protein extract was then boiled for 10 minutes in a loading buffer of 5 × SDS-PAGE. The protein was then separated by SDS-PAGE and transferred to a 0.2 µm polyvinylidene uoride membranes (PVDF). Then incubated for 2 hours at room temperature with 5% skim milk in Tris Buffered Saline, with Tween (TBST) to block the PVDF membrane. Subsequently, in 4 °C overnight incubation with the primary anti-MYOD speci c antibodies (Dilution 1:1000; ab16148; Abcam, England), anti-MYOG (1;1000; ab124800; Abcam, Cambridge, England), anti-MYH2 (Dilution 1:1000; WL02785; Wanleibio, China), anti-CDK2 (Dilution 1:1000; WL02028; Wanleibio, China), anti-PCNA (Dilution 1:500; WL03069; Wanleibio, China), anti-CyclinD1 (Dilution 1:1000; WL01435a; Wanleibio, China), anti-β-actin (1:1000; KM9001T, SungeneBiotech, China). After that, the PVDF membranes were washed with TBST buffer and then incubated at room temperature for 2 hours with the secondary antibody. The secondary antibodies were: goat anti-mouse IgG HRP (M21001S, Abmart, China), goat anti-rabbit IgG (H + L)-HRP (BA1054, Boster, China). Finally, ECL luminous liquid (DiNing, China) was used to detect the protein bands.
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