Cells were treated in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) with proteinase inhibitor cocktail (Roche, Shanghai, China) and conditionally adding phosphatase inhibitors (Sangon Biotech, Shanghai, China). The aliquots of protein were subjected to 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electrotransferred to the polyvinylidene fluoride membrane. After blocking with 5% skimmed milk and incubating with primary anti‐METTL1 (1:1500), anti‐WDR4 (1:1500, ab169526, Abcam), anti‐PI3K (1:1000), anti‐AKT (1:1000), anti‐pAKT (1:1000), anti‐mTOR (1:1000), anti‐pmTOR (1:1000), anti‐GAPDH (1:2000, 10494, Proteintech), anti‐β‐Tubulin (1:2000, 10094, Proteintech), anti‐Cyclin D1 (1:1000, ab134175, Abcam), anti‐Vimentin (1:1000, ab92547, Abcam), anti‐matrix metalloprotein 9 (MMP9) (1:1000, ab38898, Abcam), anti‐B‐cell lymphoma‐2 (Bcl‐2) (1:1000, 12789‐1‐AP, Proteintech) , anti‐Bcl‐2‐associated X protein (BAX) (1:1000, ab32503, Abcam) or anti‐phosphorylation of S6 kinase (P‐S6K) antibody (1:1000, 14485‐1‐AP, Proteintech), the membrane was then incubated with anti‐rabbit secondary antibodies (1:5000, SA00001‐2, Proteintech). Protein levels were detected using Tanon 5200 Multi intelligent imaging system (Tianneng, Shanghai, China).
Ab169526
Manufactured by Abcam
Sourced in United States
Anti-Galectin 1 antibody [EPR18773] (ab169526) is a rabbit monoclonal antibody that recognizes the Galectin 1 protein. Galectin 1 is a protein that functions in cell-cell and cell-matrix interactions.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Sangon (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Ab38898, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Cut tag kit, by Vazyme (1 mentions)
Ab271063, by Abcam (1 mentions)
Ecl system, by Vazyme (1 mentions)
Chemiscope 3300 mini imaging system, by Clinx (1 mentions)
4 protocols using ab169526
1
Western Blot Analysis of Protein Expression
2
CUT&Tag for WDR4 and DDX20 Binding
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WDR4- and DDX20-specific binding DNA fragments were detected using the CUT&Tag Kit (Vazyme). Briefly, cells (1 × 105 per sample) were captured with ConA magnetic beads and sequentially incubated with a specific antibody, secondary antibody and pA-Tn5. Antibodies against WDR4 (ab169526) were purchased from Abcam (Cambridge, MA, USA), and antibodies against DDX20 (11324-1-AP) were purchased from Proteintech (Rosemont, IL, USA). After fragmentation and extraction of DNA, the library was prepared by PCR and then sequenced on the HiSeq platform in PE150 mode. The data were analyzed using a cloud-based bioinformatics platform (Vazyme).
3
Immunohistochemical Evaluation of METTL1 and WDR4
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The TMAs were constructed by Shanghai Outdo Biotech Co., Ltd., Shanghai, China. Antibodies against METTL1 (diluted 1:5000, ab271063, Abcam, UK) and WDR4 (diluted 1:1000, ab169526, Abcam, UK) were used for IHC staining. Immunostaining was considered positive if ≥ 10% of the tumor cells were immunoreactive. Two investigators independently analyzed the IHC results using a double-blind method without knowledge of the clinical and pathological characteristics of the patients. The staining intensity and percentage of positive cells were used to evaluate METTL1 and WDR4 expression. The proportion of positive cells was classified and scored as 0–10% (0), 11–30% (1), 31–50% (2), 51–80% (3), and 81–100% (4). The staining intensity was rated as no staining (0), weak staining (1), moderate staining (2), or strong staining (3). The total score was calculated by multiplying the percentage of positively-stained cells by the staining intensity.
4
Western Blot Analysis of Protein Targets
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Protein extracts were prepared on ice using lysis buffer containing proteinase inhibitor. Samples were denatured, and proteins were separated by SDS‒PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the PVDF membranes were incubated first with primary antibodies and then with secondary antibodies. Antibodies against WDR4 (ab169526) were purchased from Abcam (Cambridge, MA, USA), and antibodies against DDX20 (11324-1-AP), Egr1 (22008-1-AP), and ARRB2 (10171-1-AP) were purchased from Proteintech (Rosemont, IL, USA). Signals were detected using an enhanced chemiluminescence (ECL) system (Vazyme, Nanjing, China), and images were acquired by a ChemiScope 3300 Mini Imaging System (CLiNX, Shanghai, China).
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