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sirna library screening c2c12 cell lines  (Addgene inc)


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    Addgene inc sirna library screening c2c12 cell lines
    Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. <t>C2C12</t> cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.
    Sirna Library Screening C2c12 Cell Lines, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sirna+library+screening+c2c12+cell+lines/10__1074_slash_jbc__ra119__008544-189-11-9?v=Addgene+inc
    Average 93 stars, based on 3 article reviews
    sirna library screening c2c12 cell lines - by Bioz Stars, 2026-07
    93/100 stars

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    1) Product Images from "A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2"

    Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

    Journal: Journal of Biological Chemistry

    doi: 10.1074/jbc.ra119.008544

    Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. C2C12 cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.
    Figure Legend Snippet: Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. C2C12 cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.

    Techniques Used: Western Blot, Library Screening, Stable Transfection, Expressing, Transfection, Fluorescence, Incubation

    Figure 4. (A–E) The indicated siRNA was expressed for 72 h in (A) cardiomyocytes or (B–E) C2C12/HA-G0S2 cells. Cells were harvested and subjected to immunoblotting. (C) Cells were solubilized and ubiquitinated proteins were enriched by TUBE pull-down assay. (D) Immunoblotting of cells that were treated with cycloheximide at the indicated times (0–120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0min) and is shown graphically. The asterisks denote statistical significance comparing siCTL- and siBAG6-treated cells. Data represent mean values ± S.D. (n=3). *P<0.05. **P<0.01. (F) Representative YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing the indicated siRNA and adenoviral Mit-ATeam for 48 h. (Scale bar, 10 µm) (G) YFP/CFP emission ratio plots of Mit-ATeam fluorescence in cardiomyocytes transfected siCTL (n=18), siBAG6 #1 (n=20) or siBAG6 #3 (n=20) during hypoxia. All of measurements were normalized to the ratio at time 0 and compared between cardiomyocytes with siCTL, siBAG6 #1 and #3 at each time point. Data represent mean values ± SEM. n.s., not significant.
    Figure Legend Snippet: Figure 4. (A–E) The indicated siRNA was expressed for 72 h in (A) cardiomyocytes or (B–E) C2C12/HA-G0S2 cells. Cells were harvested and subjected to immunoblotting. (C) Cells were solubilized and ubiquitinated proteins were enriched by TUBE pull-down assay. (D) Immunoblotting of cells that were treated with cycloheximide at the indicated times (0–120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0min) and is shown graphically. The asterisks denote statistical significance comparing siCTL- and siBAG6-treated cells. Data represent mean values ± S.D. (n=3). *P<0.05. **P<0.01. (F) Representative YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing the indicated siRNA and adenoviral Mit-ATeam for 48 h. (Scale bar, 10 µm) (G) YFP/CFP emission ratio plots of Mit-ATeam fluorescence in cardiomyocytes transfected siCTL (n=18), siBAG6 #1 (n=20) or siBAG6 #3 (n=20) during hypoxia. All of measurements were normalized to the ratio at time 0 and compared between cardiomyocytes with siCTL, siBAG6 #1 and #3 at each time point. Data represent mean values ± SEM. n.s., not significant.

    Techniques Used: Western Blot, Pull Down Assay, Fluorescence, Expressing, Transfection

    Figure 5. (A) Schematic diagram of generated mutants. (B) C2C12 cells were transfected with the indicated G0S2 mutants. After 4 h treatment with DMSO (D : 0.1%) or MG132 (MG : 10 µM), cells were harvested and subjected to immunoblot analysis. (C) C2C12/HA-G0S2 WT or E44A cells were solubilized and ubiquitinated proteins were enriched by TUBE2 pull-down assay. (D) C2C12/HA-G0S2 WT or E44A cells were treated with cycloheximide at the indicated times (0-120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0 min) and is shown graphically. The asterisks denote statistical significance comparing C2C12/HA-G0S2 WT and E44A cells. Data represent mean values ± S.D. (n=3). **P<0.01. (F) Immunoprecipitation of HA-G0S2 in HEK293T cells. Cells expressing HA-tagged G0S2 WT or E44A were harvested and immunoprecipitated with anti-HA antibody. (G) C2C12 cells expressing HA-G0S2 WT or E44A were transfected with the indicated siRNA for 48 h. After solubilizing with the buffer containing 1% Triton X-100, lysates were centrifuged and the supernatant (S) and pellet (P) fractions were analyzed by immunoblotting. The pellet contains the detergent-insoluble protein aggregates. GAPDH and vimentin are used as the detergent-soluble and -insoluble fractions, respectively.
    Figure Legend Snippet: Figure 5. (A) Schematic diagram of generated mutants. (B) C2C12 cells were transfected with the indicated G0S2 mutants. After 4 h treatment with DMSO (D : 0.1%) or MG132 (MG : 10 µM), cells were harvested and subjected to immunoblot analysis. (C) C2C12/HA-G0S2 WT or E44A cells were solubilized and ubiquitinated proteins were enriched by TUBE2 pull-down assay. (D) C2C12/HA-G0S2 WT or E44A cells were treated with cycloheximide at the indicated times (0-120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0 min) and is shown graphically. The asterisks denote statistical significance comparing C2C12/HA-G0S2 WT and E44A cells. Data represent mean values ± S.D. (n=3). **P<0.01. (F) Immunoprecipitation of HA-G0S2 in HEK293T cells. Cells expressing HA-tagged G0S2 WT or E44A were harvested and immunoprecipitated with anti-HA antibody. (G) C2C12 cells expressing HA-G0S2 WT or E44A were transfected with the indicated siRNA for 48 h. After solubilizing with the buffer containing 1% Triton X-100, lysates were centrifuged and the supernatant (S) and pellet (P) fractions were analyzed by immunoblotting. The pellet contains the detergent-insoluble protein aggregates. GAPDH and vimentin are used as the detergent-soluble and -insoluble fractions, respectively.

    Techniques Used: Generated, Transfection, Western Blot, Pull Down Assay, Immunoprecipitation, Expressing



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    sirna library screening c2c12 cell lines  (Addgene inc)
    93
    Addgene inc sirna library screening c2c12 cell lines
    Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. <t>C2C12</t> cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.
    Sirna Library Screening C2c12 Cell Lines, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sirna+library+screening+c2c12+cell+lines/10__1074_slash_jbc__ra119__008544-189-11-9?v=Addgene+inc
    Average 93 stars, based on 1 article reviews
    sirna library screening c2c12 cell lines - by Bioz Stars, 2026-07
    93/100 stars
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    Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. C2C12 cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.

    Journal: Journal of Biological Chemistry

    Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

    doi: 10.1074/jbc.ra119.008544

    Figure Lengend Snippet: Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. C2C12 cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.

    Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

    Techniques: Western Blot, Library Screening, Stable Transfection, Expressing, Transfection, Fluorescence, Incubation

    Figure 4. (A–E) The indicated siRNA was expressed for 72 h in (A) cardiomyocytes or (B–E) C2C12/HA-G0S2 cells. Cells were harvested and subjected to immunoblotting. (C) Cells were solubilized and ubiquitinated proteins were enriched by TUBE pull-down assay. (D) Immunoblotting of cells that were treated with cycloheximide at the indicated times (0–120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0min) and is shown graphically. The asterisks denote statistical significance comparing siCTL- and siBAG6-treated cells. Data represent mean values ± S.D. (n=3). *P<0.05. **P<0.01. (F) Representative YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing the indicated siRNA and adenoviral Mit-ATeam for 48 h. (Scale bar, 10 µm) (G) YFP/CFP emission ratio plots of Mit-ATeam fluorescence in cardiomyocytes transfected siCTL (n=18), siBAG6 #1 (n=20) or siBAG6 #3 (n=20) during hypoxia. All of measurements were normalized to the ratio at time 0 and compared between cardiomyocytes with siCTL, siBAG6 #1 and #3 at each time point. Data represent mean values ± SEM. n.s., not significant.

    Journal: Journal of Biological Chemistry

    Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

    doi: 10.1074/jbc.ra119.008544

    Figure Lengend Snippet: Figure 4. (A–E) The indicated siRNA was expressed for 72 h in (A) cardiomyocytes or (B–E) C2C12/HA-G0S2 cells. Cells were harvested and subjected to immunoblotting. (C) Cells were solubilized and ubiquitinated proteins were enriched by TUBE pull-down assay. (D) Immunoblotting of cells that were treated with cycloheximide at the indicated times (0–120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0min) and is shown graphically. The asterisks denote statistical significance comparing siCTL- and siBAG6-treated cells. Data represent mean values ± S.D. (n=3). *P<0.05. **P<0.01. (F) Representative YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing the indicated siRNA and adenoviral Mit-ATeam for 48 h. (Scale bar, 10 µm) (G) YFP/CFP emission ratio plots of Mit-ATeam fluorescence in cardiomyocytes transfected siCTL (n=18), siBAG6 #1 (n=20) or siBAG6 #3 (n=20) during hypoxia. All of measurements were normalized to the ratio at time 0 and compared between cardiomyocytes with siCTL, siBAG6 #1 and #3 at each time point. Data represent mean values ± SEM. n.s., not significant.

    Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

    Techniques: Western Blot, Pull Down Assay, Fluorescence, Expressing, Transfection

    Figure 5. (A) Schematic diagram of generated mutants. (B) C2C12 cells were transfected with the indicated G0S2 mutants. After 4 h treatment with DMSO (D : 0.1%) or MG132 (MG : 10 µM), cells were harvested and subjected to immunoblot analysis. (C) C2C12/HA-G0S2 WT or E44A cells were solubilized and ubiquitinated proteins were enriched by TUBE2 pull-down assay. (D) C2C12/HA-G0S2 WT or E44A cells were treated with cycloheximide at the indicated times (0-120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0 min) and is shown graphically. The asterisks denote statistical significance comparing C2C12/HA-G0S2 WT and E44A cells. Data represent mean values ± S.D. (n=3). **P<0.01. (F) Immunoprecipitation of HA-G0S2 in HEK293T cells. Cells expressing HA-tagged G0S2 WT or E44A were harvested and immunoprecipitated with anti-HA antibody. (G) C2C12 cells expressing HA-G0S2 WT or E44A were transfected with the indicated siRNA for 48 h. After solubilizing with the buffer containing 1% Triton X-100, lysates were centrifuged and the supernatant (S) and pellet (P) fractions were analyzed by immunoblotting. The pellet contains the detergent-insoluble protein aggregates. GAPDH and vimentin are used as the detergent-soluble and -insoluble fractions, respectively.

    Journal: Journal of Biological Chemistry

    Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

    doi: 10.1074/jbc.ra119.008544

    Figure Lengend Snippet: Figure 5. (A) Schematic diagram of generated mutants. (B) C2C12 cells were transfected with the indicated G0S2 mutants. After 4 h treatment with DMSO (D : 0.1%) or MG132 (MG : 10 µM), cells were harvested and subjected to immunoblot analysis. (C) C2C12/HA-G0S2 WT or E44A cells were solubilized and ubiquitinated proteins were enriched by TUBE2 pull-down assay. (D) C2C12/HA-G0S2 WT or E44A cells were treated with cycloheximide at the indicated times (0-120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0 min) and is shown graphically. The asterisks denote statistical significance comparing C2C12/HA-G0S2 WT and E44A cells. Data represent mean values ± S.D. (n=3). **P<0.01. (F) Immunoprecipitation of HA-G0S2 in HEK293T cells. Cells expressing HA-tagged G0S2 WT or E44A were harvested and immunoprecipitated with anti-HA antibody. (G) C2C12 cells expressing HA-G0S2 WT or E44A were transfected with the indicated siRNA for 48 h. After solubilizing with the buffer containing 1% Triton X-100, lysates were centrifuged and the supernatant (S) and pellet (P) fractions were analyzed by immunoblotting. The pellet contains the detergent-insoluble protein aggregates. GAPDH and vimentin are used as the detergent-soluble and -insoluble fractions, respectively.

    Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

    Techniques: Generated, Transfection, Western Blot, Pull Down Assay, Immunoprecipitation, Expressing