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Experimental Validation. ( A – C ) Western blot detection of muscle atrophy-related proteins Atrogin-1 and <t>MuRF1.</t> Atrogin-1, Significant differences by one-way ANOVA ( P < 0.0001) and Tukey’s test: N vs. M *** P = 0.0002, M vs. APS-H *** P = 0.0002, n = 3. MuRF1, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.044, M vs. APS-H * P = 0.492, n = 3. ( D ) Electron microscope images of the control group (Group N), model group (Group M), and high-dose APS group (Group APS-H). The left images show muscle tissue observed at 5000 × magnification, high-contrast mode, with an accelerating voltage of 80.0 kV. The right images show mitochondria and autophagosomes observed under the same conditions at 20,000 × magnification. Blue arrows indicate mitochondria, and yellow arrows indicate autophagosomes. ( E – I ) Western blot detection of autophagy-related proteins BNIP3, LC3-I/II, GABARAPL1, and SQSTM1/p62. BNIP3, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.0035, M vs. APS-H ** P = 0.003, n = 3. LC3-I/II, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.005, M vs. APS-H * P = 0.0399, n = 3. GABARAPL1, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0204, M vs. APS-H * P = 0.049, n = 3. p62, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.0027, M vs. APS-H * P = 0.03, n = 3. ( J – L ) Western blot analysis of the transcriptional regulator FOXO3 and p-FOXO3. FOXO3, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0152, M vs. APS-H * P = 0.0263, n = 3. p-FOXO3, Significant differences by one-way ANOVA ( P < 0.001) and Tukey’s test: N vs. M *** P = 0.0005, M vs. APS-H * P = 0.0347, n = 3. ( M – P ) Western blot detection of eNOS and nNOS. eNOS, Significant differences by one-way ANOVA ( P < 0.001) and Tukey’s test: N vs. M *** P = 0.0002, M vs. APS-H ** P = 0.0034, n = 3. nNOS, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0116, M vs. APS-H * P = 0.0384, n = 3.
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Experimental Validation. ( A – C ) Western blot detection of muscle atrophy-related proteins Atrogin-1 and MuRF1. Atrogin-1, Significant differences by one-way ANOVA ( P < 0.0001) and Tukey’s test: N vs. M *** P = 0.0002, M vs. APS-H *** P = 0.0002, n = 3. MuRF1, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.044, M vs. APS-H * P = 0.492, n = 3. ( D ) Electron microscope images of the control group (Group N), model group (Group M), and high-dose APS group (Group APS-H). The left images show muscle tissue observed at 5000 × magnification, high-contrast mode, with an accelerating voltage of 80.0 kV. The right images show mitochondria and autophagosomes observed under the same conditions at 20,000 × magnification. Blue arrows indicate mitochondria, and yellow arrows indicate autophagosomes. ( E – I ) Western blot detection of autophagy-related proteins BNIP3, LC3-I/II, GABARAPL1, and SQSTM1/p62. BNIP3, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.0035, M vs. APS-H ** P = 0.003, n = 3. LC3-I/II, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.005, M vs. APS-H * P = 0.0399, n = 3. GABARAPL1, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0204, M vs. APS-H * P = 0.049, n = 3. p62, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.0027, M vs. APS-H * P = 0.03, n = 3. ( J – L ) Western blot analysis of the transcriptional regulator FOXO3 and p-FOXO3. FOXO3, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0152, M vs. APS-H * P = 0.0263, n = 3. p-FOXO3, Significant differences by one-way ANOVA ( P < 0.001) and Tukey’s test: N vs. M *** P = 0.0005, M vs. APS-H * P = 0.0347, n = 3. ( M – P ) Western blot detection of eNOS and nNOS. eNOS, Significant differences by one-way ANOVA ( P < 0.001) and Tukey’s test: N vs. M *** P = 0.0002, M vs. APS-H ** P = 0.0034, n = 3. nNOS, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0116, M vs. APS-H * P = 0.0384, n = 3.

Journal: Scientific Reports

Article Title: Integrated transcriptomics and metabolomics studies reveal the therapeutic effects of Astragalus polysaccharides on cancer cachexia muscle atrophy

doi: 10.1038/s41598-025-11342-x

Figure Lengend Snippet: Experimental Validation. ( A – C ) Western blot detection of muscle atrophy-related proteins Atrogin-1 and MuRF1. Atrogin-1, Significant differences by one-way ANOVA ( P < 0.0001) and Tukey’s test: N vs. M *** P = 0.0002, M vs. APS-H *** P = 0.0002, n = 3. MuRF1, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.044, M vs. APS-H * P = 0.492, n = 3. ( D ) Electron microscope images of the control group (Group N), model group (Group M), and high-dose APS group (Group APS-H). The left images show muscle tissue observed at 5000 × magnification, high-contrast mode, with an accelerating voltage of 80.0 kV. The right images show mitochondria and autophagosomes observed under the same conditions at 20,000 × magnification. Blue arrows indicate mitochondria, and yellow arrows indicate autophagosomes. ( E – I ) Western blot detection of autophagy-related proteins BNIP3, LC3-I/II, GABARAPL1, and SQSTM1/p62. BNIP3, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.0035, M vs. APS-H ** P = 0.003, n = 3. LC3-I/II, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.005, M vs. APS-H * P = 0.0399, n = 3. GABARAPL1, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0204, M vs. APS-H * P = 0.049, n = 3. p62, Significant differences by one-way ANOVA ( P < 0.01) and Tukey’s test: N vs. M ** P = 0.0027, M vs. APS-H * P = 0.03, n = 3. ( J – L ) Western blot analysis of the transcriptional regulator FOXO3 and p-FOXO3. FOXO3, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0152, M vs. APS-H * P = 0.0263, n = 3. p-FOXO3, Significant differences by one-way ANOVA ( P < 0.001) and Tukey’s test: N vs. M *** P = 0.0005, M vs. APS-H * P = 0.0347, n = 3. ( M – P ) Western blot detection of eNOS and nNOS. eNOS, Significant differences by one-way ANOVA ( P < 0.001) and Tukey’s test: N vs. M *** P = 0.0002, M vs. APS-H ** P = 0.0034, n = 3. nNOS, Significant differences by one-way ANOVA ( P < 0.05) and Tukey’s test: N vs. M * P = 0.0116, M vs. APS-H * P = 0.0384, n = 3.

Article Snippet: Primary antibodies used for the experiments: Anti-beta Tubulin (M1305-2, 1:5000, HUABIO, Hangzhou, China); Anti-Fbx32 (Atriogin-1) (ET7109-25, 1:2000, HUABIO, Hangzhou, China); Anti-BNIP3 (ET1704-08, 1:500, HUABIO, Hangzhou, China); Anti-LC3B (ET1701-65, 1:1000, HUABIO, Hangzhou, China); Anti-Phospho-FOXO3a (S253) (ET1609-49, 1:1000, HUABIO, Hangzhou, China); Anti-nNOS (ET1609-61, 1:1000, HUABIO, Hangzhou, China); SQSTM1 (p62) (HA721171, 1:5000, HUABIO, Hangzhou, China); TRIM63 (Murf1) (55,456–1-AP, 1:1000, Proteintech, Wuhan, China); GABARAPL1 (11,010–1-AP, 1:1000, Proteintech, Wuhan, China); eNOS (27,120–1-AP, 1:500, Proteintech, Wuhan, China); FOXO3A (66,428–1-Ig, 1:2000, Proteintech, Wuhan, China).

Techniques: Biomarker Discovery, Western Blot, Microscopy, Control

Transcriptomic mechanism map. ( A ) Autophagy-lysosomal pathway and mitochondrial autophagy. The transition of LC3-I to LC3-II promotes the formation of autophagosomal membranes. GABARAPL1-I/II are functionally analogous to LC3-I/II. The p62 receptor labels ubiquitinated proteins and mitochondria, while the BNIP3 receptor directly targets mitochondria. The autophagosomal membrane encapsulates cargo such as proteins and mitochondria, which are then recognized by lysosomes for degradation. ( B ) Ubiquitin–proteasome pathway. MuRF1 and Atrogin-1 are both E3 ubiquitin ligases that tag proteins for further ubiquitination. These ubiquitinated proteins are recognized by the proteasome, leading to their degradation. ( C ) FOXO3 is involved in the transcriptional regulation of associated proteins.

Journal: Scientific Reports

Article Title: Integrated transcriptomics and metabolomics studies reveal the therapeutic effects of Astragalus polysaccharides on cancer cachexia muscle atrophy

doi: 10.1038/s41598-025-11342-x

Figure Lengend Snippet: Transcriptomic mechanism map. ( A ) Autophagy-lysosomal pathway and mitochondrial autophagy. The transition of LC3-I to LC3-II promotes the formation of autophagosomal membranes. GABARAPL1-I/II are functionally analogous to LC3-I/II. The p62 receptor labels ubiquitinated proteins and mitochondria, while the BNIP3 receptor directly targets mitochondria. The autophagosomal membrane encapsulates cargo such as proteins and mitochondria, which are then recognized by lysosomes for degradation. ( B ) Ubiquitin–proteasome pathway. MuRF1 and Atrogin-1 are both E3 ubiquitin ligases that tag proteins for further ubiquitination. These ubiquitinated proteins are recognized by the proteasome, leading to their degradation. ( C ) FOXO3 is involved in the transcriptional regulation of associated proteins.

Article Snippet: Primary antibodies used for the experiments: Anti-beta Tubulin (M1305-2, 1:5000, HUABIO, Hangzhou, China); Anti-Fbx32 (Atriogin-1) (ET7109-25, 1:2000, HUABIO, Hangzhou, China); Anti-BNIP3 (ET1704-08, 1:500, HUABIO, Hangzhou, China); Anti-LC3B (ET1701-65, 1:1000, HUABIO, Hangzhou, China); Anti-Phospho-FOXO3a (S253) (ET1609-49, 1:1000, HUABIO, Hangzhou, China); Anti-nNOS (ET1609-61, 1:1000, HUABIO, Hangzhou, China); SQSTM1 (p62) (HA721171, 1:5000, HUABIO, Hangzhou, China); TRIM63 (Murf1) (55,456–1-AP, 1:1000, Proteintech, Wuhan, China); GABARAPL1 (11,010–1-AP, 1:1000, Proteintech, Wuhan, China); eNOS (27,120–1-AP, 1:500, Proteintech, Wuhan, China); FOXO3A (66,428–1-Ig, 1:2000, Proteintech, Wuhan, China).

Techniques: Membrane, Ubiquitin Proteomics