antibodies against rich1  (Santa Cruz Biotechnology)

Journal: Molecular Medicine Reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: RICH1 is downregulated in ISO- or Ang II-treated H9c2 cells. Western blotting results of RICH1 and α-tubulin in (A) ISO- and (B) Ang II-treated cells. H9c2 cells were treated with phosphate-buffered saline in the control group, and 60 µM ISO or 3 µM Ang II in the experimental groups, and total protein was extracted for western blotting. Statistical evaluation of protein expression in cells treated with (C) 60 µM ISO (n=8 experimental repeats/group) and (D) 3 µM Ang II (n=10 experimental repeats/group) normalized to α-tubulin in each group. Data were analyzed using unpaired Student's t-test and presented as the mean ± SEM. **P<0.01. ISO, isoproterenol; Ang II, angiotensin II; RICH1, Rho GTPase-activating protein interacting with CIP4 homologs protein 1.

Article Snippet: The blots were then incubated with primary antibodies against RICH1 (1:500; cat. no. sc-514438; Santa Cruz Biotechnology, Inc.) and α-tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4°C, followed by anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL001A; Biosharp Life Sciences) or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL003A; Biosharp Life Sciences) for 1 h at room temperature.

Techniques: Western Blot, Saline, Control, Expressing

Journal: Molecular Medicine Reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: Overexpression of RICH1 attenuates ISO- or Ang II-induced cardiomyocyte hypertrophy. (A) Protein expression levels of RICH1 in H9c2 cells transfected with pCDNA3.1-RICH1 plasmids were assessed by western blotting and (B) RICH1 expression was normalized to α-tubulin (n=8 experimental repeats/group). Data were analyzed by unpaired Student's t-test and presented as the mean ± SEM. H9c2 cells were stained with Acti-stain™ 488 phalloidin and captured using an Olympus FV3000 confocal microscope. Overexpression of RICH1 can inhibit the enlargement of CSA induced by (C) ISO or (D) Ang II. Scale bar, 40 µm. CSA quantification in response to (E) ISO (n=53/group) and (F) Ang II (n=39 number of cells assessed/group). This experiment was repeated three times. Data were analyzed by one-way ANOVA with Tukey's post hoc test and are presented as the mean ± SD. Reverse transcription-quantitative PCR was used to assess the expression levels of the cardiac hypertrophy-related genes (G and L) Nppa , (H and K) Nppb and (I and L) Myh7 , which were normalized to Gapdh . Overexpression of RICH1 can inhibit the upregulation of cardiac hypertrophy marker genes induced by (G-I) ISO or (J-L) Ang II (n=5-6 experimental repeats/group). Data were analyzed using one-way ANOVA with Tukey's post hoc test and are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. CSA, cell surface area; ISO, isoproterenol; Ang II, angiotensin II; OE, overexpression; Rho GTPase-activating protein interacting with CIP4 homologs protein 1.

Article Snippet: The blots were then incubated with primary antibodies against RICH1 (1:500; cat. no. sc-514438; Santa Cruz Biotechnology, Inc.) and α-tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4°C, followed by anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL001A; Biosharp Life Sciences) or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL003A; Biosharp Life Sciences) for 1 h at room temperature.

Techniques: Over Expression, Expressing, Transfection, Western Blot, Staining, Microscopy, Reverse Transcription, Real-time Polymerase Chain Reaction, Marker

Journal: Molecular Medicine Reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: siRNA-directed RICH1 knockdown exacerbates ISO- or Ang II-induced cardiomyocyte hypertrophy. (A) Protein expression levels of RICH1 in H9c2 cells transfected with siRICH1 and siNC were assessed by western blotting, and (B) RICH1 expression was normalized to α-tubulin (n=6 experimental repeats/group). Data were analyzed using one-way ANOVA with Tukey's post hoc test and are presented as the mean ± SEM. H9c2 cells were stained with Acti-stain™ 488 phalloidin and captured under an Olympus FV3000 confocal microscope. Knockdown of RICH1 enhanced the enlargement of CSA induced by (C) ISO or (D) Ang II. Scale bar, 40 µm. CSA quantification in response to (E) ISO (n=48 number of cells assessed/group) and (F) Ang II (n=55 number of cells assessed/group). This experiment was repeated three times. Data were analyzed using one-way ANOVA with Tukey's post hoc test and are presented as the mean ± SD. Expression levels of the cardiac hypertrophy-related genes (G and J) Nppa , (H and K) Nppb and (I and L) Myh7 treated with (G-I) ISO or (J-L) Ang II were assessed by reverse transcription-quantitative PCR and normalized to Gapdh (n=4-6 experimental repeats/group). Data were analyzed using one-way ANOVA with Tukey's post hoc test and are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. CSA, cell surface area; ISO, isoproterenol; Ang II, angiotensin II; NC, negative control; si, small interfering; ns, not significant; RICH1, Rho GTPase-activating protein interacting with CIP4 homologs protein 1.

Article Snippet: The blots were then incubated with primary antibodies against RICH1 (1:500; cat. no. sc-514438; Santa Cruz Biotechnology, Inc.) and α-tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4°C, followed by anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL001A; Biosharp Life Sciences) or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL003A; Biosharp Life Sciences) for 1 h at room temperature.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Staining, Microscopy, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Molecular Medicine Reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: Schematic diagram of molecular mechanisms by which RICH1 mediates cardiomyocyte hypertrophy. Overexpression of RICH1 inhibited cardiomyocyte hypertrophy induced by ISO and Ang II. Active Rho GTPase proteins can positively promote the expression of cardiac hypertrophy-related genes, Nppa, Nppb and Myh7 . RICH1 can induce the inactivation of Rho. Under the stimulation of ISO and Ang II, RICH1 is downregulated. Therefore, more Rho GTPase proteins remain active and enhance the expression of cardiomyocyte hypertrophy-related genes. When RICH1 is overexpressed, Rho GTPase proteins are inactivated and cardiac hypertrophy-related genes are downregulated. ISO, isoproterenol; Ang II, angiotensin II; RICH1, Rho GTPase-activating protein interacting with CIP4 homologs protein.

Article Snippet: The blots were then incubated with primary antibodies against RICH1 (1:500; cat. no. sc-514438; Santa Cruz Biotechnology, Inc.) and α-tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4°C, followed by anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL001A; Biosharp Life Sciences) or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL003A; Biosharp Life Sciences) for 1 h at room temperature.

Techniques: Over Expression, Expressing

Journal: Molecular medicine reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy.

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: Figure 1. RICH1 is downregulated in ISO‑ or Ang II‑treated H9c2 cells. Western blotting results of RICH1 and α‑tubulin in (A) ISO‑ and (B) Ang II‑treated cells. H9c2 cells were treated with phosphate‑buffered saline in the control group, and 60 µM ISO or 3 µM Ang II in the experimental groups, and total protein was extracted for western blotting. Statistical evaluation of protein expression in cells treated with (C) 60 µM ISO (n=8 experimental repeats/group) and (D) 3 µM Ang II (n=10 experimental repeats/group) normalized to α‑tubulin in each group. Data were analyzed using unpaired Student's t‑test and presented as the mean ± SEM. **P<0.01. ISO, isoproterenol; Ang II, angiotensin II; RICH1, Rho GTPase‑activating protein interacting with CIP4 homologs protein 1.

Article Snippet: The blots were then incubated with primary anti‐ bodies against ricH1 (1:500; cat. no. sc‐514438; Santa cruz Biotechnology, inc.) and α‐tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4 ̊C, followed by anti‐mouse horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl001a; Biosharp life Sciences) or anti‐rabbit horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl003a; Biosharp life Sciences) for 1 h at room temperature.

Techniques: Western Blot, Saline, Control, Expressing

Journal: Molecular medicine reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy.

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: Figure 2. Overexpression of RICH1 attenuates ISO‑ or Ang II‑induced cardiomyocyte hypertrophy. (A) Protein expression levels of RICH1 in H9c2 cells transfected with pCDNA3.1‑RICH1 plasmids were assessed by western blotting and (B) RICH1 expression was normalized to α‑tubulin (n=8 experimental repeats/group). Data were analyzed by unpaired Student's t‑test and presented as the mean ± SEM. H9c2 cells were stained with Acti‑stain™ 488 phalloidin and captured using an Olympus FV3000 confocal microscope. Overexpression of RICH1 can inhibit the enlargement of CSA induced by (C) ISO or (D) Ang II. Scale bar, 40 µm. CSA quantification in response to (E) ISO (n=53/group) and (F) Ang II (n=39 number of cells assessed/group). This experiment was repeated three times. Data were analyzed by one‑way ANOVA with Tukey's post hoc test and are presented as the mean ± SD. Reverse transcription‑quantitative PCR was used to assess the expression levels of the cardiac hypertrophy‑related genes (G and L) Nppa, (H and K) Nppb and (I and L) Myh7, which were normal‑ ized to Gapdh. Overexpression of RICH1 can inhibit the upregulation of cardiac hypertrophy marker genes induced by (G‑I) ISO or (J‑L) Ang II (n=5‑6 experimental repeats/group). Data were analyzed using one‑way ANOVA with Tukey's post hoc test and are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. CSA, cell surface area; ISO, isoproterenol; Ang II, angiotensin II; OE, overexpression; Rho GTPase‑activating protein interacting with CIP4 homologs protein 1.

Article Snippet: The blots were then incubated with primary anti‐ bodies against ricH1 (1:500; cat. no. sc‐514438; Santa cruz Biotechnology, inc.) and α‐tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4 ̊C, followed by anti‐mouse horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl001a; Biosharp life Sciences) or anti‐rabbit horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl003a; Biosharp life Sciences) for 1 h at room temperature.

Techniques: Over Expression, Expressing, Transfection, Western Blot, Staining, Microscopy, Marker

Journal: Molecular medicine reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy.

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: Figure 3. siRNA‑directed RICH1 knockdown exacerbates ISO‑ or Ang II‑induced cardiomyocyte hypertrophy. (A) Protein expression levels of RICH1 in H9c2 cells transfected with siRICH1 and siNC were assessed by western blotting, and (B) RICH1 expression was normalized to α‑tubulin (n=6 experimental repeats/group). Data were analyzed using one‑way ANOVA with Tukey's post hoc test and are presented as the mean ± SEM. H9c2 cells were stained with Acti‑stain™ 488 phalloidin and captured under an Olympus FV3000 confocal microscope. Knockdown of RICH1 enhanced the enlargement of CSA induced by (C) ISO or (D) Ang II. Scale bar, 40 µm. CSA quantification in response to (E) ISO (n=48 number of cells assessed/group) and (F) Ang II (n=55 number of cells assessed/group). This experiment was repeated three times. Data were analyzed using one‑way ANOVA with Tukey's post hoc test and are presented as the mean ± SD. Expression levels of the cardiac hypertrophy‑related genes (G and J) Nppa, (H and K) Nppb and (I and L) Myh7 treated with (G‑I) ISO or (J‑L) Ang II were assessed by reverse transcription‑quantitative PCR and normalized to Gapdh (n=4‑6 experimental repeats/group). Data were analyzed using one‑way ANOVA with Tukey's post hoc test and are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. CSA, cell surface area; ISO, isoproterenol; Ang II, angiotensin II; NC, negative control; si, small interfering; ns, not significant; RICH1, Rho GTPase‑activating protein interacting with CIP4 homologs protein 1.

Article Snippet: The blots were then incubated with primary anti‐ bodies against ricH1 (1:500; cat. no. sc‐514438; Santa cruz Biotechnology, inc.) and α‐tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4 ̊C, followed by anti‐mouse horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl001a; Biosharp life Sciences) or anti‐rabbit horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl003a; Biosharp life Sciences) for 1 h at room temperature.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Staining, Microscopy, Negative Control

Journal: Molecular medicine reports

Article Title: RICH1 is a novel key suppressor of isoproterenol‑ or angiotensin II‑induced cardiomyocyte hypertrophy.

doi: 10.3892/mmr.2024.13193

Figure Lengend Snippet: Figure 4. Schematic diagram of molecular mechanisms by which RICH1 mediates cardiomyocyte hypertrophy. Overexpression of RICH1 inhibited cardio‑ myocyte hypertrophy induced by ISO and Ang II. Active Rho GTPase proteins can positively promote the expression of cardiac hypertrophy‑related genes, Nppa, Nppb and Myh7. RICH1 can induce the inactivation of Rho. Under the stimulation of ISO and Ang II, RICH1 is downregulated. Therefore, more Rho GTPase proteins remain active and enhance the expression of cardiomyocyte hypertrophy‑related genes. When RICH1 is overexpressed, Rho GTPase proteins are inactivated and cardiac hypertrophy‑related genes are downregulated. ISO, isoproterenol; Ang II, angiotensin II; RICH1, Rho GTPase‑activating protein interacting with CIP4 homologs protein.

Article Snippet: The blots were then incubated with primary anti‐ bodies against ricH1 (1:500; cat. no. sc‐514438; Santa cruz Biotechnology, inc.) and α‐tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4 ̊C, followed by anti‐mouse horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl001a; Biosharp life Sciences) or anti‐rabbit horseradish peroxidase‐conjugated secondary antibodies (1:50,000; cat. no. Bl003a; Biosharp life Sciences) for 1 h at room temperature.

Techniques: Over Expression, Expressing

Journal: Cell death & disease

Article Title: Wdr4 promotes cerebellar development and locomotion through Arhgap17-mediated Rac1 activation.

doi: 10.1038/s41419-022-05442-z

Figure Lengend Snippet: Fig. 6 Wdr4 promotes the ubiquitination and degradation of Arhgap17 to activate Rac1. A The Volcano plot of proteins identified by LC- MS/MS from GNPs isolated from the P7 Wdr4 A-cKO; Ai14 and control cerebella. Differentially expressed proteins (p < 0.05 and fold change > 1.5) are marked in blue (for up-regulated) and green (for down-regulated). Among the upregulated proteins, those with a known anti-proliferative function are marked in red, orange or yellow with their names. Data were from 3 cerebella in each group and were analyzed using pairwise-ratio-based, Student’s t-test by the Proteome Discoverer 2.3 software. B, C Immunoprecipitation analysis for the interaction between exogenous (B) or endogenous (C) Wdr4 with indicated proteins. D–E Western blot (D) and RT-qPCR (E) analyses for the expression of Arhgap17 protein or Arhgap17 mRNA in N2a cells expressing control or Wdr4 shRNAs. Data in (E) were from four repeats in each group and analyzed using one-way ANOVA post hoc Dunnett’s test, p = 0.3302 in shWdr4#1 v.s. shCtrl, and 0.1855 in shWdr4#2 v.s. shCtrl. Data are represented as individual points and mean. F Western blot analysis for Arhgap17 and Gli2 expression in the purified GNPs from the P7 Wdr4 A-cKO and control cerebella. G, H Western blot analysis for Arhgap17 expression in N2a cells stably expressing Wdr4 (G) or Wdr4 shRNAs (H) and treated with 1 μM MG132 for 16 h. I Western blot analysis for Arhgap17 expression in N2a cells stably expressing Wdr4 and treated with 100 μg/ml CHX for indicated time points. The levels of Arhgap17 were normalized to the 0 h time point in the control or Wdr4-expressing group, respectively, and indicated at the bottom. J, K In vivo ubiquitination assay using N2a cells (J) or N2a cells expressing Wdr4 shRNAs (K) and transfected with indicated constructs. L Rac1 activity assay using N2a cells expressing Wdr4 shRNAs or transfected with Flag-Arhgap17. The western blot results were quantified using ImageJ software. The protein levels were normalized first to the Gapdh protein level in each group and then to the corresponding control groups, and expressed as fold changes at the bottom. All western blot analyses were done at least twice.

Article Snippet: Plasmids encoding mouse Wdr4 (#MC201825, NM_021322) and Arhgap17 (#MR210303, NM_001122643) were purchased from OriGene Technologies.

Techniques: Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Isolation, Control, Software, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, Stable Transfection, In Vivo, Transfection, Construct, Activity Assay