Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet:

Article Snippet: The primary antibodies and dilutions used were: mouse monoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780 ); rabbit anti-SARS-CoV-1 3CL pro antibody (1:2000, Rockland, 200-401-A51, RRID: AB_828457 ); rabbit polyclonal anti-RPAP1 antibody (1:1,000, Proteintech, 15138-1-AP, RRID: AB_2301137 ); mouse monoclonal anti-PTBP1 antibody (1:500, Biolegend, 630101, 3H7, RRID: AB_2171285 ); rabbit polyclonal anti-MAP4K5 antibody (1:1,000, Cusabio, CSB-PA013440DSR2HU, RRID: AB_2892084 ); rabbit polyclonal anti-CREB1 antibody (1:1,000, Abclonal, A11989, RRID: AB_2758916 ); rabbit polyclonal anti-YAP1 antibody (1:1,000, Abclonal, A11430, RRID: AB_2758556 ); rabbit polyclonal anti-FYCO1 antibody (1:1,000, Cusabio, CSB-PA866262LA01HU, RRID: AB_2892085 ); rabbit polyclonal anti-FAF1 antibody (1:1,000, Abclonal, A2921, RRID: AB_2764739 ); goat polyclonal anti-Gal8 antibody (1:400, R&D Systems, AF1305, RRID: AB_2137229 ); rabbit polyclonal anti-KPNA3 (IMA4) antibody (1:1,000, Abclonal, A8347, RRID: AB_2770124 ); rabbit polyclonal anti-NUP107 antibody (1:1,000, Abclonal, A13110, RRID: AB_2759959 ); mouse monoclonal anti-IRS2 antibody (1:300, R&D Systems, MAB6347, 676415, RRID: AB_10992928 ); mouse monoclonal anti-FLAG M2 antibody (1:10,000, Sigma, F3165, RRID: AB_259529 ); mouse monoclonal anti-β-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-β-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371 ); rabbit monoclonal anti-β-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528 ).

Techniques: Virus, Recombinant, Sequencing, Fluorescence, Modification, Protease Inhibitor, Polymer, Staining, Blocking Assay, Binding Assay, Activity Assay, Western Blot, Synthesized, Software, Control, Targeted Proteomics, Microscopy, Spectrophotometry, Mass Spectrometry

Journal: Endocrinology

Article Title: Essential Role of IGFIR in the Onset of Male Brown Fat Thermogenic Function: Regulation of Glucose Homeostasis by Differential Organ-Specific Insulin Sensitivity

doi: 10.1210/en.2015-1623

Figure Lengend Snippet: Antibody Table

Article Snippet: Phospho-Akt (Thr308) (C31E5E) Rabbit mAb Cell Signaling Technology, #2965 rabbit; monoclonal 1:1000 AKT Endogenous levels of total Akt1, Akt2 and Akt3 proteins Akt Antibody Cell Signaling Technology, #9272 rabbit; polyclonal 1:1000 BMP7 Synthetic peptide corresponding to residues in Human BMP7 Anti-BMP7 antibody [EPR5897] Epitomics, #5626-1 rabbit; monoclonal 1:500 HSP60 HSP60 (mouse), (recombinant) Enzo Life Sciences, ADI-SPP-741 mouse; 1:1000 β2-adrenergic receptor Against a peptide mapping at the C terminus of B2-AR of mouse origin β2-AR (M-20) Santa Cruz Biotechnology, sc-570 rabbit; polyclonal 1:1000 UCP-1 Synthetic peptide conjugated to KLH, corresponding to amino acids 145–159 of Human UCP-1, with N-terminal cysteine added Anti-UCP-1 antibody Abcam, ab 10983 rabbit; polyclonal IHC, 1:500 IRS1 Carboxy-terminal 14 amino acid peptide ([C]YASINFQKQPEDRQ) of rat liver IRS1 Anti-IRS1 Millipore #06-248 rabbit; polyclonal 1:500 IRS2 GST fusion protein containing amino acids 976–1094 of mouse IRS2 Anti-IRS2 Millipore #06-506 rabbit; polyclonal 1:500 β-actin N-terminal end of the β-isoform of actin Anti-β-actin, clone AC-74 Sigma-Aldrich, A2228 mouse; monoclonal 1:5000 Tubulin C-terminal end of the α-tubulin isoform (amino acids 426–430) Anti-α-tubulin, clone DM1A Sigma-Aldrich, T6199 mouse; monoclonal 1:5000 GAPDH Anti-GAPDH antibody, clone GAPDH 71.1 Sigma-Aldrich, G8795 mouse; monoclonal 1:5000 Open in a separate window Antibody Table

Techniques: Sequencing, Recombinant

Journal: Molecular and Cellular Biology

Article Title: A Regulatory Role for MicroRNA 33* in Controlling Lipid Metabolism Gene Expression

doi: 10.1128/MCB.01714-12

Figure Lengend Snippet: Posttranscriptional regulation of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 by miR-33a and miR-33a* in Huh7 cells. (A) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NYFC, and RIP140 in Huh7 cells transfected with a control mimic (CM), miR-33a mimic, or miR-33a* mimic. (B) Western blot analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 in Huh7 cells transfected with CM, miR-33a, or miR-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. a.u., arbitrary units. (C) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NYFC, and RIP140 in Huh7 cells transfected with a control inhibitor (CI), inhibitor of miR-33a (Inh-33a), or inhibitor of miR-33a* (Inh-33a*). (D) Western blot analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 in Huh7 cells transfected with CI, Inh-33a, or Inh-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. In panels A to D, the data are means ± SEM and representative of ≥3 experiments in triplicate. *, P ≤ 0.05.

Article Snippet: A rabbit polyclonal antibody against IRS2 was purchased from Bethyl, and a rabbit polyclonal antibody against RIP140 was acquired from Santa Cruz.

Techniques: Quantitative RT-PCR, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Regulatory Role for MicroRNA 33* in Controlling Lipid Metabolism Gene Expression

doi: 10.1128/MCB.01714-12

Figure Lengend Snippet: Predicted lipid metabolism target genes for miR-33a*

Article Snippet: A rabbit polyclonal antibody against IRS2 was purchased from Bethyl, and a rabbit polyclonal antibody against RIP140 was acquired from Santa Cruz.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: A Regulatory Role for MicroRNA 33* in Controlling Lipid Metabolism Gene Expression

doi: 10.1128/MCB.01714-12

Figure Lengend Snippet: miR-33a* specifically targets the 3′ UTR of human genes Npc1, Crot, Irs2, Src1, Src3, and Nyfc, while miR-33b* specifically targets the 3′ UTR of human Nfyc. (A through F) Luciferase reporter activity in COS7 cells transfected with control mimic (CM) or miR-33a* mimic and the human or mouse (A) Npc1, (B) Crot, (C) Irs2, (D) Src1, (E) Src3, or (F) Nfyc 3′ UTRs containing the indicated point mutations (PM) in the miR-33a* target sites. (G) Luciferase reporter activity in COS7 cells transfected with control mimic (CM) or miR-33b* mimic and the human Nfyc 3′ UTR containing the indicated point mutations (PM) in the miR-33b* target site. (H) qRT-PCR analysis of CROT and NPC1 in Huh7 cells transfected with the control mimic (CM), miR-33a, or miR-33a* after Ago2 immunoprecipitation (Ago2-IP). In panels A to G, the data are expressed as mean percentages of 3′-UTR activity of CM ± SEM and are representative of ≥3 experiments in triplicate. *, P ≤ 0.05. n.s., not significant. In panel H, the data are means ± SEM and representative of 2 experiments in duplicate.

Article Snippet: A rabbit polyclonal antibody against IRS2 was purchased from Bethyl, and a rabbit polyclonal antibody against RIP140 was acquired from Santa Cruz.

Techniques: Luciferase, Activity Assay, Transfection, Control, Quantitative RT-PCR, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: A Regulatory Role for MicroRNA 33* in Controlling Lipid Metabolism Gene Expression

doi: 10.1128/MCB.01714-12

Figure Lengend Snippet: Both arms of the miR-33 duplex contribute to the posttranscriptional regulation of target gene expression. (A to B) Huh7 cells were transduced with a lentivirus encoding the miR-33a precursor (pre-miR-33) or empty vector control. Transduction efficiency was confirmed 48 h after culturing by measuring GFP expression using fluorescence microscopy (A) and flow cytometry (B). (C) qRT-PCR analysis of miR-33a (top panel) and miR-33a* (bottom panel) in empty vector-transduced or pre-miR-33-transduced Huh7 cells. (D) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, RIP140, and NFYC in Huh7 cells transduced with pre-miR-33. Values were compared to those from empty vector-transduced cells. (E) qRT-PCR analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, RIP140, and NFYC in pre-miR-33-transduced Huh7 cells transfected with a control inhibitor (CI), inhibitor of miR-33a (Inh-miR-33a), or inhibitor of miR-33a* (Inh-miR-33a*). (F) Western blot analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 in pre-miR-33-transduced Huh7 cells transfected with CI, Inh-miR-33a, or Inh-miR-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. FS, forward scatter. In panels A to F, the data are means ± SEM and representative of ≥2 experiments in triplicate. *, P ≤ 0.05.

Article Snippet: A rabbit polyclonal antibody against IRS2 was purchased from Bethyl, and a rabbit polyclonal antibody against RIP140 was acquired from Santa Cruz.

Techniques: Targeted Gene Expression, Transduction, Plasmid Preparation, Control, Expressing, Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Transfection, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Regulatory Role for MicroRNA 33* in Controlling Lipid Metabolism Gene Expression

doi: 10.1128/MCB.01714-12

Figure Lengend Snippet: Proposed model of miR-33 arm-specific processing and target gene regulation. miR-33a* and -b* work collectively with their sister strands, miR-33a and -b, to boost intracellular cholesterol and fatty acid levels by balancing the posttranscriptional repression of genes involved in cellular cholesterol efflux (ABCA1 and NPC1), fatty acid oxidation (CROT, CPT1a, HADHB, and AMPK), glucose metabolism (SIRT6 and IRS2), and the transcriptional regulation of lipid metabolism (SRC1, SRC3, NFYC, and RIP140). Pol II, polymerase II.

Article Snippet: A rabbit polyclonal antibody against IRS2 was purchased from Bethyl, and a rabbit polyclonal antibody against RIP140 was acquired from Santa Cruz.

Techniques: