Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: Effects of Arg and Leu on the location of mTOR on LAMP2-positive lysosomal clusters, activation of mTORC1 and expression of SLC38A9 and SLC36A1 in C2C12 cells. ( A ) C2C12 cells were pretreated without serum for 15 h and without amino acids for 3 h then cultured for 10 min in a special medium including the no amino acids group (−AA), the only Arg group (+Arg), and the only Leu group (+Leu). Cells were stained with anti-mTOR (A2445, Abclonal Technology, Wuhan, China) or anti-LAMP2. Scale bar: 10 µM. ( B ) Ratio of mTOR to LAMP2 (%). To assess mTOR translocation, the numbers of mTOR and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. ( C ) Immunoblotting analysis of protein samples from ( A ) with anti-mTOR, anti-phospho-mTOR, anti-S6K, anti-phospho-S6K, anti-SLC38A9, anti-SLC36A1, or anti-β-Actin antibody. ( D ) Densitometric analysis of the immunodetection of mTOR relative to β-Actin loading control. ( E ) Densitometric analysis of the immunodetection of phospho-mTOR relative to β-Actin loading control. ( F ) Densitometric analysis of the immunodetection of p70S6K relative to β-Actin loading control. ( G ) Densitometric analysis of the immunodetection of phospho-p70S6K relative to β-Actin loading control. ( H ) Densitometric analysis of the immunodetection of SLC38A9 relative to β-Actin loading control. ( I ) Densitometric analysis of the immunodetection of SLC36A1 relative to β-Actin loading control. ( J ) The mRNA levels of SLC38A9 and SLC36A1 were analyzed by qRT-PCR. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activation Assay, Expressing, Cell Culture, Staining, Translocation Assay, Software, Western Blot, Immunodetection, Control, Quantitative RT-PCR

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: The effects of SLC38A9 or SLC36A1 overexpression and inhibition on mTOR phosphorylation in C2C12 cells. ( A ) The mRNA expression of SLC38A9 after overexpression of SLC38A9. ( B ) The mRNA expression of SLC38A9 after inhibition of SLC38A9. ( C ) The mRNA expression of SLC36A1 after overexpression of SLC36A1. ( D ) The mRNA expression of SLC36A1 after inhibition of SLC36A1. ( E ) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct or siRNA to overexpress or knockdown SLC38A9. Immunoblotting analysis of protein samples with anti-SLC38A9, anti-mTOR, anti-phospho-mTOR, or anti-β-Actin antibody. ( F ) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct or siRNA to overexpress or knockdown SLC36A1. Immunoblotting analysis of protein samples with anti-SLC36A1, anti-mTOR, anti-phospho-mTOR, or anti-β-Actin antibody. ( G ) Densitometric analysis of the immunodetection of SLC38A9, mTOR or phospho-mTOR relative to β-Actin loading control from ( E ). ( H ) Densitometric analysis of the immunodetection of SLC36A1, mTOR or phospho-mTOR relative to β-Actin loading control from ( F ). Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Over Expression, Inhibition, Phospho-proteomics, Expressing, Transfection, Construct, Knockdown, Western Blot, Immunodetection, Control

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: SLC38A9 increases SLC36A1 expression and promotes the translocation of SLC36A1 to LAMP2-positive lysosomal clusters. ( A ) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct to overexpress SLC38A9. The mRNA level of SLC36A1 was analyzed by qRT-PCR. ( B ) C2C12 cells were transfected with siRNA to knockdown SLC38A9. The mRNA level of SLC36A1 was analyzed by qRT-PCR. ( C ) Immunoblotting analysis of protein samples from ( A , B ) with anti-SLC36A1 or anti-β-Actin antibody. ( D ) Densitometric analysis of the immunodetection of SLC36A1 relative to β-Actin loading control. ( E ) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct or siRNA to overexpress or knockdown SLC38A9 and stained with anti-SLC36A1 or anti-LAMP2. Scale bar: 10µM. ( F ) Ratio of SLC36A1 to LAMP2 (%). To assess SLC36A1 translocation, the numbers of SLC36A1 and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Translocation Assay, Transfection, Construct, Quantitative RT-PCR, Knockdown, Western Blot, Immunodetection, Control, Staining, Software

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: SLC36A1 increases SLC38A9 expression and promotes the translocation of SLC38A9 to LAMP2-positive lysosomal clusters. ( A ) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct to overexpress SLC36A1. The mRNA level of SLC38A9 was analyzed by qRT-PCR. ( B ) C2C12 cells were transfected with siRNA to knockdown SLC36A1. The mRNA level of SLC38A9 was analyzed by qRT-PCR. ( C ) Immunoblotting analysis of protein samples from ( A , B ) with anti-SLC38A9 or anti-β-Actin antibody. ( D ) Densitometric analysis of the immunodetection of SLC38A9 relative to β-Actin loading control. ( E ) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct or siRNA to overexpress or knockdown SLC36A1 and stained with anti-SLC38A9 or anti-LAMP2. Scale bar: 10µM. ( F ) Ratio of SLC38A9 to LAMP2 (%). To assess SLC38A9 translocation, the numbers of SLC38A9 and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Translocation Assay, Transfection, Construct, Quantitative RT-PCR, Knockdown, Western Blot, Immunodetection, Control, Staining, Software

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: SLC38A9 and SLC36A1 interact. ( A ) SLC38A9 was associated with SLC36A1 at the endogenous level as detected by Co-IP: anti-SLC36A1 (sc-161150, Santa Cruz) was used for pull-down, and anti-SLC38A9 (ab81687, Abcam) and anti-SLC36A1 (sc-368553, Santa Cruz) were used for detection. ( B ) Interaction of SLC38A9 and SLC36A1: C2C12 cells were transfected with SLC38A9 or SLC36A1 in the pCMV-HA vector, and the lysates were prepared and subjected to HA immunoprecipitation followed by immunoblotting for the indicated proteins. ( C ) C2C12 cells were pretreated without serum for 15 h, and without amino acids for 3 h, then cultured for 10 min in a special medium including the no amino acids group (−AA), the only Arg group (+Arg), the only Leu group (+Leu). The interaction of SLC38A9 and SLC36A1 was detected by Co-IP as ( A ). ( D ) Densitometric analysis of the immunodetection of SLC38A9 relative to input control or SLC36A1. Values are the mean ± SEM; n = 3; * p < 0.05.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Cell Culture, Immunodetection, Control