rabbit monoclonal anti-stk11 (clone 27d10)  (Cell Signaling Technology Inc)

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: The m6A mRNA methylation regulates AMPK activity in Leydig cells (LCs). (A) m6A levels in LCs were assessed by immunofluorescence staining. Fluorescence intensity was presented as means ± SEM (n = 3). *P < 0.05 vs. post-natal day 10 (D10) group. (B) Protein levels of m6A regulatory enzymes in LCs at various developmental stages were examined by western blotting and quantitatively analyzed. Data were presented as means ± SEM (n = 3). *P < 0.05 vs. D10 group. (C) Relative m6A levels were measured by ELISA-based m6A quantitative analyses. *P < 0.05 vs. D10 group. TM3 cells were treated with HsCG for indicated times. (D) The expression of m6A regulatory enzymes were examined by western blotting and quantitatively analyzed. *P < 0.05 vs. the control cells. (E) Relative m6A levels were measured by ELISA-based m6A quantitative analyses. *P < 0.05 vs. the control cells. (F) The expression of both HSD3B and m6A levels in LCs at 6 h was examined by immunofluorescence staining. Fluorescence intensity was presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells. (G) TM3 cells were transfected with increasing doses of pcDNA3.1-Mettl14 vector (O/E Mettl14; O/E, overexpression) for 36 h followed by HsCG treatment for 6 h. The cell extracts were subjected to western blotting and quantitative analysis. Data are presented as the means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the HsCG-treated cells. (H) TM3 cells were transfected with siRNA targeting Alkbh5 (si-Alkbh5) and cultured for 36 h followed by HsCG treatment for 6 h. The cell extracts were subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the HsCG-treated cells

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: Methylation, Activity Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Over Expression, Cell Culture

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: Upregulated m6A levels were observed in Leydig cells (LCs) from azoospermia or oligospermia patients with lowered levels of serum testosterone. (A) The expression of LC3B in LCs at 6 h was examined by immunofluorescence staining, and the average LC3B puncta number per cell was quantified. (B) m6A levels in LCs were assessed by immunofluorescence assay. (C and D) The protein levels of METTL14 and ALKBH5 in LCs were determined by immunofluorescence staining. Data are presented as means ± SEM. *P < 0.05

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: m6A mRNA methylation impacts the expression of regulators of the AMPK pathway. (A) TM3 cells were transfected with shRNA targeting Mettl14 (sh-Mettl14) followed by compound C treatment for 6 h. The cell extracts were subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the sh-Mettl14-treated cells. (B) TM3 cells were transfected with increasing doses of pcDNA3.1-Mettl14 vector (O/E Mettl14; O/E, overexpression). The cell extracts were subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells. (C) TM3 cells were transfected with sh-Mettl14 before treatment with siRNA targeting Camkk2 (si-Camkk2), and cell lysate was subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the sh-Mettl14-treated cells. (D) TM3 cells were transfected with the pcDNA3.1-Alkbh5 (O/E Alkbh5) vector before treatment with si-Camkk2, and cell lysate was subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the O/E Alkbh5-treated cells. (E) TM3 cells were transfected with sh-Mettl14 before transfection with pcDNA3.1-Ppm1a (O/E Ppm1a), and cell lysate was subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the sh-Mettl14-treated cells. (F) TM3 cells were transfected with pcDNA3.1-Alkbh5 (O/E Alkbh5) before transfection with pcDNA3.1-Ppm1a (O/E Ppm1a), and cell lysate was subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the O/E Alkbh5-treated cells

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: Methylation, Expressing, Transfection, shRNA, Western Blot, Plasmid Preparation, Over Expression

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: m6A induces the decay of the Camkk2 transcript in Leydig cells (LCs). TM3 cells were treated with indicated siRNAs. (A and B) Cell lysates were subjected to western blotting and quantitative analysis. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells. (C) mRNA levels of target genes were measured by qRT-PCR. *P < 0.05 vs. the control cells. (D) YTHDF1/YTHDF2 was immunoprecipitated, followed by qRT-PCR for assessing the association of the indicated transcripts with each protein. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the IgG group. (E) Schematic representation of the position of m6A motifs with Camkk2 transcript. (F) Abundance of Camkk2 transcript among mRNA immunoprecipitated with anti-m6A antibody was measured by qRT-PCR and normalized to input. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the IgG group. (G) Abundance of Camkk2 transcript among mRNA immunoprecipitated with anti-m6A antibody from cells with or without transfection of shRNA targeting Alkbh5 (sh-Alkbh5) was measured by qRT-PCR. Data are presented as means ± SEM (n = 3). *p < 0.05. (H) Camkk2-3ʹ-UTR of the wild-type or containing an m6A consensus sequence mutant (A to G) was fused with a luciferase reporter. (I) Luciferase activity of Camkk2-3ʹ-UTR was measured and normalized to Renilla luciferase activity. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells. (J) TM3 cells pre-treated with indicated siRNAs were treated with actinomycin D (5 μg/mL) for 2 h, followed by measurement of Camkk2 and Ppm1a mRNA levels at indicated times. *P < 0.05 vs. the control cells

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: Western Blot, Quantitative RT-PCR, Immunoprecipitation, Transfection, shRNA, Sequencing, Mutagenesis, Luciferase, Activity Assay

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: m6A triggers the translation of the Ppm1a transcript. (A and B) TM3 cells with or without transfection of shRNA targeting Mettl14 (sh-Mettl14) were pre-treated with cycloheximide (CHX, 10 μg/mL) for 3 h followed by exposure to HsCG for indicated times. Expression levels of PPM1A were examined by western blotting and quantitatively analyzed. (C and D) TM3 cells with or without transfection of shRNA targeting Alkbh5 (sh-Alkbh5) were pre-treated with MG-132 (2 μM) for 1.5 h followed by exposure to HsCG for 6 h. Expression levels of PPM1A were examined by western blotting and quantitatively analyzed. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the MG-132-treated cells. (E) Schematic representation of the position of m6A motifs with Ppm1a transcript. (F) Abundance of the Ppm1a transcripts among mRNA immunoprecipitated with anti-m6A antibody was measured by qRT-PCR. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the IgG group. (G) Abundance of the Ppm1a transcript among mRNA immunoprecipitated with anti-m6A antibody from cells with or without transfection of sh-Alkbh5 was measured by qRT-PCR. Data are presented as means ± SEM (n = 3). *P < 0.05. (H) Ppm1a-3ʹ-UTR of the wild-type or containing an m6A consensus sequence mutant (A to G) were fused with a luciferase reporter. (I and J) Luciferase activity of Camkk2-3ʹ-UTR was measured and normalized to Renilla luciferase activity. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells. (K) Schematic representation of mutation in pcDNA3.1-Ppm1a-CDS-3ʹUTR is shown. (L) TM3 cells were transfected with sh-Mettl14 followed by transfection with indicated vectors, and cell lysate was subjected to western blotting

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: Transfection, shRNA, Expressing, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Sequencing, Mutagenesis, Luciferase, Activity Assay

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: HsCG induces transcription of Alkbh5 in Leydig cells (LCs). (A) TM3 cells were treated with HsCG for indicated times, and mRNA levels of Mettl14 and Alkbh5 were analyzed by qRT-PCR. *P < 0.05 vs. the control cells. (B and C) TM3 cells were pre-treated with cycloheximide (CHX, 10 μg/mL) for 3 h followed by exposure to HsCG for indicated times, and protein levels of METTL14 were assessed by western blotting and quantitatively analyzed. *P < 0.05 vs. the CHX-treatment cells. (D) TM3 cells were pre-treated with MG-132 (2 μM) for 1.5 h followed by exposure to HsCG for 6 h. Expression levels of METTL14 were examined by western blotting and quantitatively analyzed. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells. (E) Potential transcription factor binding sites in the Alkbh5 promoter region were predicted. (F) TM3 cells were treated with HsCG for indicated times. Both cytoplasmic and nuclear proteins were isolated, followed by western blotting and quantitative analysis. ACTB and LMNB1 served as cytosolic and nuclear markers, respectively. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells. (G and H) TM3 cells were transfected with siRNA targeting Cebpb (si-Cebpb) or si-Tfeb and cultured for 36 h followed by exposure to HsCG for 6 h. The expression of ALKBH5 was examined by western blotting and quantitatively analyzed. Data are presented as means ± SEM (n = 3). *P < 0.05 vs. the control cells; #P < 0.05 vs. the HsCG-treated cells. (I and J) TM3 cells were transfected with Alkbh5 promoter constructs harboring indicated mutations of TEFB binding sites (I) or CEBPB binding sites (J). Luciferase activity was measured, and data were presented as means ± SEM (n = 3). *P < 0.05. An “NS” denotes no statistically significant difference between the testing groups

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Binding Assay, Isolation, Transfection, Cell Culture, Construct, Luciferase, Activity Assay

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: Reduced nuclear translocation of TFEB and CEBPB is observed in aged Leydig cells (LCs). Enrichment of TFEB (A) and CEBPB (B) in the nucleus of LCs was determined by immunofluorescence analysis. The merging of the red and green fluorescence signals was quantified with Image J software. D10, post-natal days 10; D540, post-natal days 540. (C) A model for the critical link between METTL14/ALKBH5 and autophagy in HsCG-treated LCs is proposed. HsCG reduces METTL14 expression and increases ALKBH5 expression by suppressing the stability of METTL14 and enhancing Alkbh5 transcription, respectively. Both downregulation of METTL14 and upregulation of ALKBH5 lead to reduced m6A mRNA methylation levels. Reduction of m6A levels alleviates m6A-mediated Ppm1a translation and upregulates CAMKK2 expression by retarding m6A-mediated decay of the transcript, contributing to activation of PRKAA2 and subsequently autophagy initiation

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: Translocation Assay, Immunofluorescence, Fluorescence, Software, Expressing, Methylation, Activation Assay

Journal: Autophagy

Article Title: m 6 A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

doi: 10.1080/15548627.2020.1720431

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: Working dilution Abcam Rabbit anti-LC3B ab48394 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-SQSTM1 ab56416 WB, 1:1000 Abcam Rabbit anti-METTL3 ab195352 WB, 1:2000 IF, 1: 1000 Abcam Mouse anti-FTO ab92981 WB, 1:1000 Boster Rabbit anti-ACTB MO1263-2 WB,1:500 Cell Signaling Technology Rabbit anti-ULK1 6439 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S757 14,202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-ULK1 S555 5869 WB,1:1000 Cell Signaling Technology Rabbit anti-PRKAA2 5831 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PRKAA2 T172 50,081 WB,1:1000 IF, 1:50 Cell Signaling Technology Rabbit anti-MTOR 2983 WB,1:1000 Cell Signaling Technology Rabbit anti-p-MTOR S2448 5536 WB,1:1000 Cell Signaling Technology Rabbit anti-RPS6KB1 9202 WB,1:1000 Cell Signaling Technology Rabbit anti-p-RPS6KB1 9205 WB,1:1000 Cell Signaling Technology Rabbit anti-PPM1A 3549 WB,1:1000 Cell Signaling Technology Rabbit anti-SMAD3 9523 WB,1:1000 Cell Signaling Technology Rabbit anti-FOS 2250 WB,1:1000 Cell Signaling Technology Rabbit anti-p-PIK3C3/VPS34 13,857 WB,1:1000 Santa Cruz Biotechnology Mouse anti-HSD3B sc-100,466 IF,1:50 Santa Cruz Biotechnology Mouse anti-SP1 sc-420 WB,1:500 Santa Cruz Biotechnology Mouse anti-FOXM1 sc-376,471 WB,1:500 Santa Cruz Biotechnology Mouse anti-CEBPB sc-7962 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-TFEB sc-166,736 WB,1:500 ChIP,1:50 Santa Cruz Biotechnology Mouse anti-NR5A1 sc-393,592 WB,1:500 Santa Cruz Biotechnology Mouse anti-MYC sc-40 WB,1:500 Santa Cruz Biotechnology Mouse anti-LMNB1 sc-374,015 WB,1:500 Santa Cruz Biotechnology Mouse anti-EEF2 sc-166,415 WB,1:500 Santa Cruz Biotechnology Mouse anti-EIF3A sc-376,651 WB,1:500 Santa Cruz Biotechnology Mouse anti-PIK3C3/VPS34 sc-365,404 WB,1:500 Proteintech Rabbit anti-CAMKK2 11,549-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-STK11/LKB1 10,746-1-AP WB,1:1000 Proteintech Rabbit anti-ALKBH5 16,837-1-AP WB,1:1000 IF,1:50 Proteintech Rabbit anti-YTHDF1 17,479-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-YTHDF2 24,744-1-AP WB,1:1000 Co-IP, 1:50 RIP, 1:50 Proteintech Rabbit anti-RELA 10,745-1-AP WB,1:1000 Sigma Rabbit anti-METTL14 HPA038002 WB,1:2000 IF,1:100 Synaptic Systems Rabbit anti-m 6 A 202,003 IF,1:200 Open in a separate window Primary antibodies used in this study Plasmid and siRNA transfection Gene sequences coding for Mettl14 and Alkbh5 were cloned into the pcDNA3.1 vector (Invitrogen, V79020).

Techniques: