Western Blot Analysis of Protein Interactions

Total cell extracts were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Western blot was performed as previously described [17 (link)]. Primary antibodies against KEAP1 (1:500, sc-514,914, USA) and ARIH1 (1:500, sc-514,551) were purchased from Santa Cruz (MD, USA). Primary antibodies against Cleaved-caspase-3 (#9664), caspase-3 (#9662), PARP (#9542), cleaved-PARP (#9541), vimentin (#5741), ubiquitin (#8240), Flag (#14,793), HA(#3724), GFP (#2555), FTO (#31,687), METTL3 (#86,132), METTL14 (#51,104), YTHDF2 (#80,014), STAT1 (#9172),p-STAT1 (#8826), P65 (#8242), p-p65 (#3033), RIG-I (#3743), IRF7 (#4920), IRF3 (#11,904), p-IRF7 (#24,129), p-IRF3 (#4947), PCNA (#2586), SOX2 (#23,064), ABCG2 (#4477), NAONG (#4903), GAPDH (#2118), and β-actin (#4967) were purchased from Cell Signaling Technology. Densitometry analysis of Western blots was performed using Image J and the band densities were normalized to GAPDH. The control was set to 1 for plotted graphs. Protein identification was performed by LC-MS/MS. Briefly, 50 µg proteins was subjected to SDS-PAGE and the silver stained protein bands were excised and analyzed by LC-MS/MS (BGI, Shenzhen, China.

Free full text: Click here

Zhang Y., Liu Z., Zhong Z., Ji Y., Guo H., Wang W, & Chen C. (2024). A tumor suppressor protein encoded by circKEAP1 inhibits osteosarcoma cell stemness and metastasis by promoting vimentin proteasome degradation and activating anti-tumor immunity. Journal of Experimental & Clinical Cancer Research : CR, 43, 52.

Publication 2024

protease and phosphatase inhibitor cocktail KEAP1 Cleaved-caspase-3 caspase-3 cleaved-PARP vimentin ubiquitin Flag METTL3 METTL14 YTHDF2 STAT1 p-STAT1 p-IRF7 p-IRF3 PCNA ABCG2 GAPDH β-actin

Corresponding Organization : Cancer Hospital of Shantou University Medical College

Other organizations : First Affiliated Hospital of Shantou University Medical College

Top 5 similar protocols

Variable analysis

independent variables

KEAP1 antibody
ARIH1 antibody

dependent variables

Cleaved-caspase-3
Caspase-3
Cleaved-PARP
Vimentin
Ubiquitin
METTL3
METTL14
YTHDF2
STAT1
P-STAT1
P-p65
RIG-I
P-IRF7
P-IRF3
ABCG2
NAONG

control variables

GAPDH
β-actin

positive controls

None specified

negative controls

None specified

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!