Journal: Signal Transduction and Targeted Therapy

Article Title: MARCH3 negatively regulates IL-3-triggered inflammatory response by mediating K48-linked polyubiquitination and degradation of IL-3Rα

doi: 10.1038/s41392-021-00834-7

Figure Lengend Snippet: MARCH2 and MARCH3 negatively regulates IL-3-triggered signaling. a Effects of overexpression of the MARCH proteins on IL-3Rα level. Human embryonic kidney 293 cells (4 × 10 5 ) were transfected with expression plasmids for the indicated FLAG-tagged proteins, HA-tagged IL-3Rα and GFP for 24 h before immunoblots for detection of the indicated proteins. b Knockout efficiency of the MARCH proteins. Human erythroleukemia TF-1 cells were transduced with control, g MARCH2 , g MARCH3 , or g MARCH8 by lentiviral-mediated gene transfer. The knockout cells were analyzed by immunoblots for detection of the indicated proteins. c Effects of MARCH2, 3, 8-deficiency on IL-3Rα levels. The indicated TF-1 cells (2 × 10 7 ) were starved overnight and then stimulated with IL-3 (20 ng/mL) for the indicated times. Cell lysates were immunoprecipitated with anti-IL-3Rα (Santa Cruz Biotechnology, SC-455). The immunoprecipitates were analyzed by immunoblots with anti-IL-3Rα (MYBioSource, MBS2544022). Lysates were analyzed by immunoblots with anti-β-actin. d Effects of MARCH2, 3, 8-deficiency on IL-3-induced transcription of downstream genes. The indicated TF-1 cells (5 × 10 5 ) were starved overnight and then left untreated or treated with IL-3 (10 ng/mL) for the indicated times before qPCR analysis. Data shown are means ± SEM from one representative experiment performed in triplicate. ** P < 0.01, *** P < 0.001; ns, not significant. e Effects of IL-3 on the mRNA and protein levels of MARCH2, 3, 8. TF-1 cells (5 × 10 5 ) were starved overnight and then stimulated with IL-3 (20 ng/mL) for the indicated times before qPCR and immunoblotting analysis for detection of the indicated mRNAs and proteins. Data shown are means ± SEM from one representative experiment performed in triplicate. All the experiments were repeated for at least two times with similar results

Article Snippet: Recombinant human GM-CSF (PeproTech, 300-03) and IL-3 (PeproTech, 200-03), murine Il-3 (PeproTech, 213-13) and Il-34 (Sino Biological, 50055-M08H), M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase inhibitor (Thermo Scientific, EO0382), RNAiso plus (Takara Bio, 9109), SYBR Green mix (Bio-Rad, 172-5274); MG132 (MCE, HY-13259), polybrene (Millipore, TR-1003-G); RPMI 1640 medium (Gibco, C11875500BT), Dulbecco’s Modified Eagle Medium (Gibco, C11995500BT), FBS (Cellmax, SA211.02), penicillin/streptomycin (Gibo, 15140-122); ELISA kits for murine Tnf-α (BioLegend, 430904), Il-6 (BioLegend, 431304), Il-1β (Boster bio, EK0394); PNGase F (NEW ENGLAND BioLabs, P0704S); Mouse monoclonal antibodies against β-actin (Sigma-Aldrich, A2228), FLAG ® M2 (Sigma-Aldrich, F3165), HA (BioLegend, 901515), Myc (Cell Signaling Technology, 2276), phosphor-p38 T180/Y182 (Cell Signaling Technology, 9216), human IL-3Rα (for Co-IP) (Santa Cruz Biotechnology, SC-455); Rabbit antibodies against phospho-STAT5 Y694/699 (Cell Signaling Technology, 4322), phospho-AKT S473 (Cell Signaling Technology, 4060), p38 (Cell Signaling Technology, 8690), STAT5 (Cell Signaling Technology, 94205), AKT (Cell Signaling Technology, 4691), human IL-3Rα (for immunoblot) (MYBioSource, MBS2544022), murine Il-3rα (MYBioSource, MBS2542745), murine March2 (Abcam, ab220292), MARCH8 (Proteintech, 14119-1-AP), K48-linkage specific polyubiquitin (Abcam, ab140601); HRP-conjugated mouse anti-HA (Sigma-Aldrich, H6533); Goat anti-mouse IgG (Jackson Immuno Research, 115-035-003) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Knock-Out, Transduction, Immunoprecipitation

Journal: Signal Transduction and Targeted Therapy

Article Title: MARCH3 negatively regulates IL-3-triggered inflammatory response by mediating K48-linked polyubiquitination and degradation of IL-3Rα

doi: 10.1038/s41392-021-00834-7

Figure Lengend Snippet: March3-deficiency promotes Il-3-induced signaling in BMDMs. a Effects of March2, 3-deficiency on Il-3rα levels. Wild-type, March2 −/− and March3 −/− bone marrow-derived macrophages (BMDMs) (1 × 10 6 ) were left untreated or treated with Il-3 (50 ng/mL) for the indicated times before immunoblotting analysis for detection of Il-3rα and β-actin. b Effects of March2, 3-deficiency on Il-3-induced phosphorylation of signaling components. The indicated BMDMs (1 × 10 6 ) were left untreated or treated with Il-3 (25 ng/mL) for the indicated times before immunoblotting analysis for detection of the indicated proteins. c Effects of March2, 3-deficiency on Il-3-induced transcription of downstream genes. Wild-type, March2 −/− or March3 −/− BMDMs (1 × 10 6 ) were left untreated or treated with Il-3 (25 ng/mL) for the indicated times before qPCR analysis. Data shown are means ± SEM from one representative experiment performed in triplicate. * P < 0.05, *** P < 0.001; ns, not significant. All the experiments were repeated for at least two times with similar results

Article Snippet: Recombinant human GM-CSF (PeproTech, 300-03) and IL-3 (PeproTech, 200-03), murine Il-3 (PeproTech, 213-13) and Il-34 (Sino Biological, 50055-M08H), M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase inhibitor (Thermo Scientific, EO0382), RNAiso plus (Takara Bio, 9109), SYBR Green mix (Bio-Rad, 172-5274); MG132 (MCE, HY-13259), polybrene (Millipore, TR-1003-G); RPMI 1640 medium (Gibco, C11875500BT), Dulbecco’s Modified Eagle Medium (Gibco, C11995500BT), FBS (Cellmax, SA211.02), penicillin/streptomycin (Gibo, 15140-122); ELISA kits for murine Tnf-α (BioLegend, 430904), Il-6 (BioLegend, 431304), Il-1β (Boster bio, EK0394); PNGase F (NEW ENGLAND BioLabs, P0704S); Mouse monoclonal antibodies against β-actin (Sigma-Aldrich, A2228), FLAG ® M2 (Sigma-Aldrich, F3165), HA (BioLegend, 901515), Myc (Cell Signaling Technology, 2276), phosphor-p38 T180/Y182 (Cell Signaling Technology, 9216), human IL-3Rα (for Co-IP) (Santa Cruz Biotechnology, SC-455); Rabbit antibodies against phospho-STAT5 Y694/699 (Cell Signaling Technology, 4322), phospho-AKT S473 (Cell Signaling Technology, 4060), p38 (Cell Signaling Technology, 8690), STAT5 (Cell Signaling Technology, 94205), AKT (Cell Signaling Technology, 4691), human IL-3Rα (for immunoblot) (MYBioSource, MBS2544022), murine Il-3rα (MYBioSource, MBS2542745), murine March2 (Abcam, ab220292), MARCH8 (Proteintech, 14119-1-AP), K48-linkage specific polyubiquitin (Abcam, ab140601); HRP-conjugated mouse anti-HA (Sigma-Aldrich, H6533); Goat anti-mouse IgG (Jackson Immuno Research, 115-035-003) were purchased from the indicated companies.

Techniques: Derivative Assay, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: MARCH3 negatively regulates IL-3-triggered inflammatory response by mediating K48-linked polyubiquitination and degradation of IL-3Rα

doi: 10.1038/s41392-021-00834-7

Figure Lengend Snippet: MARCH3 mediates K48-linked polyubiquitination of IL-3Rα. a Endogenous MARCH3 is associated with IL-3Rα. Human erythroleukemia TF-1 cells (2 × 10 7 ) were starved overnight and then left untreated or treated with IL-3 (20 ng/mL) for the indicated times. Cell lysates were immunoprecipitated with control mouse IgG (IgG) or anti-IL-3Rα as indicated. The immunoprecipitates were analyzed by immunoblots with rabbit anti-IL-3Rα and mouse anti-MARCH3 as indicated. Lysates were analyzed by immunoblots with rabbit anti-IL-3Rα, mouse anti-MARCH3 and anti-β-actin to determine levels of the respective endogenous proteins. b MARCH3 but not its E3 ligase-inactive mutants mediates polyubiquitination of IL-3Rα. Human embryonic kidney 293 cells (2 × 10 6 ) were transfected with expression plasmids for FLAG-tagged IL-3Rα, Myc-tagged wild-type ubiquitin (Ub) and the indicated HA-tagged wild-type or mutant MARCH3 for 20 h. Cell lysates were immunoprecipitated with anti-FLAG. The immunoprecipitates were denatured and then re-immunoprecipitated with anti-FLAG. The final immunoprecipitates and cell lysates were analyzed by immunoblots for detection of the indicated proteins. c MARCH3-deficient TF-1 cells were reconstituted with wild-type MARCH3 and its E3 ligase-inactive mutants by lentiviral-mediated gene transfer. The reconstituted cells (5 × 10 5 ) were starved overnight and then stimulated with IL-3 (10 ng/mL) for 1 h before qPCR analysis for mRNA levels of the indicated genes. Data shown are means ± SEM from one representative experiment performed in triplicate. d Linkage types of polyubiquitination of IL-3Rα. HEK293 cells (2 × 10 6 ) were transfected with expression plasmids for FLAG-tagged IL-3Rα, HA-tagged MARCH3 and HA-tagged wild-type ubiquitin (Ub) or its mutants for 20 h. Cell lysates were immunoprecipitated with anti-FLAG. The immunoprecipitates were denatured and then re-immunoprecipitated with anti-FLAG. The final immunoprecipitates and cell lysates were analyzed by immunoblots for detection of the indicated proteins. e Effects of MARCH3-deficiency on IL-3-induced K48-linked polyubiquitination of IL-3Rα. Control and MARCH3-deficient TF-1 (2 × 10 7 ) cells were starved overnight and then left untreated or stimulated with IL-3 (20 ng/mL) for the indicated times. Cell lysates were then immunoprecipitated with anti-IL-3Rα. The immunoprecipitates were denatured and then re-immunoprecipitated with anti-IL-3Rα. The final immunoprecipitates were analyzed by immunoblots with anti-Ub-K48 or anti-IL-3Rα. Cell lysates were analyzed by immunoblots for detection of the indicated proteins. f Effects of inhibitors on MARCH3-mediated degradation of IL-3Rα. HEK293 cells (4 × 10 5 ) were transfected with expression plasmids for FLAG-tagged IL-3Rα, Myc-tagged MARCH3 and HA-tagged β-actin as indicated for 12 h, and then treated with the lysosomal inhibitor NH 4 Cl (5 mM) or the proteasome inhibitor MG132 (0.5 μM) for 6 h before immunoblotting analysis with anti-FLAG, anti-Myc and anti-HA respectively. All the experiments were repeated twice with similar results

Article Snippet: Recombinant human GM-CSF (PeproTech, 300-03) and IL-3 (PeproTech, 200-03), murine Il-3 (PeproTech, 213-13) and Il-34 (Sino Biological, 50055-M08H), M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase inhibitor (Thermo Scientific, EO0382), RNAiso plus (Takara Bio, 9109), SYBR Green mix (Bio-Rad, 172-5274); MG132 (MCE, HY-13259), polybrene (Millipore, TR-1003-G); RPMI 1640 medium (Gibco, C11875500BT), Dulbecco’s Modified Eagle Medium (Gibco, C11995500BT), FBS (Cellmax, SA211.02), penicillin/streptomycin (Gibo, 15140-122); ELISA kits for murine Tnf-α (BioLegend, 430904), Il-6 (BioLegend, 431304), Il-1β (Boster bio, EK0394); PNGase F (NEW ENGLAND BioLabs, P0704S); Mouse monoclonal antibodies against β-actin (Sigma-Aldrich, A2228), FLAG ® M2 (Sigma-Aldrich, F3165), HA (BioLegend, 901515), Myc (Cell Signaling Technology, 2276), phosphor-p38 T180/Y182 (Cell Signaling Technology, 9216), human IL-3Rα (for Co-IP) (Santa Cruz Biotechnology, SC-455); Rabbit antibodies against phospho-STAT5 Y694/699 (Cell Signaling Technology, 4322), phospho-AKT S473 (Cell Signaling Technology, 4060), p38 (Cell Signaling Technology, 8690), STAT5 (Cell Signaling Technology, 94205), AKT (Cell Signaling Technology, 4691), human IL-3Rα (for immunoblot) (MYBioSource, MBS2544022), murine Il-3rα (MYBioSource, MBS2542745), murine March2 (Abcam, ab220292), MARCH8 (Proteintech, 14119-1-AP), K48-linkage specific polyubiquitin (Abcam, ab140601); HRP-conjugated mouse anti-HA (Sigma-Aldrich, H6533); Goat anti-mouse IgG (Jackson Immuno Research, 115-035-003) were purchased from the indicated companies.

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Mutagenesis

Journal: Signal Transduction and Targeted Therapy

Article Title: MARCH3 negatively regulates IL-3-triggered inflammatory response by mediating K48-linked polyubiquitination and degradation of IL-3Rα

doi: 10.1038/s41392-021-00834-7

Figure Lengend Snippet: MARCH3 mediates K48-linked polyubiquitination of IL-3Rα at K377. a MARCH3 catalyzes K48-linked polyubiquitination of IL-3Rα at K377. Human embryonic kidney 293 cells (2 × 10 6 ) were transfected with the indicated plasmids for 20 h. Cell lysates were then immunoprecipitated with anti-FLAG. The immunoprecipitates were denatured and then re-immunoprecipitated with anti-FLAG. The final immunoprecipitates and cell lysates were analyzed by immunoblots with anti-HA or anti-FLAG. HA-Ub (K48O), HA-tagged ubiquitin with all lysine residues mutated to arginine except K48. b IL-3Rα K377R has increased ability than IL-3Rα in mediating IL-3-triggered signaling. IL-3Rα-deficient human erythroleukemia TF-1 cells were reconstituted with wild-type IL-3Rα and IL-3Rα K377R by lentiviral-mediated gene transfer. The reconstituted TF-1 cells (5 × 10 5 ) were starved overnight and stimulated with IL-3 (10 ng/mL) for 1 h before qPCR analysis for mRNA levels of the indicated genes. Data shown are means ± SEM from one representative experiment performed in triplicate. c Sequence alignment of the cytoplasmic domains of human and murine IL-3Rα. The sequences are corresponding to aa326-378 of human IL-3Rα and aa356-396 of murine Il-3rα. d March3 but not March2 catalyzes K48-linked polyubiquitination of Il-3rα at K357. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids for 20 h. Cell lysates were then immunoprecipitated with anti-FLAG. The immunoprecipitates were denatured and then re-immunoprecipitated with anti-FLAG. The final immunoprecipitates and cell lysates were analyzed by immunoblots with anti-FLAG or anti-HA. All the experiments were repeated for at least two times with similar results

Article Snippet: Recombinant human GM-CSF (PeproTech, 300-03) and IL-3 (PeproTech, 200-03), murine Il-3 (PeproTech, 213-13) and Il-34 (Sino Biological, 50055-M08H), M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase inhibitor (Thermo Scientific, EO0382), RNAiso plus (Takara Bio, 9109), SYBR Green mix (Bio-Rad, 172-5274); MG132 (MCE, HY-13259), polybrene (Millipore, TR-1003-G); RPMI 1640 medium (Gibco, C11875500BT), Dulbecco’s Modified Eagle Medium (Gibco, C11995500BT), FBS (Cellmax, SA211.02), penicillin/streptomycin (Gibo, 15140-122); ELISA kits for murine Tnf-α (BioLegend, 430904), Il-6 (BioLegend, 431304), Il-1β (Boster bio, EK0394); PNGase F (NEW ENGLAND BioLabs, P0704S); Mouse monoclonal antibodies against β-actin (Sigma-Aldrich, A2228), FLAG ® M2 (Sigma-Aldrich, F3165), HA (BioLegend, 901515), Myc (Cell Signaling Technology, 2276), phosphor-p38 T180/Y182 (Cell Signaling Technology, 9216), human IL-3Rα (for Co-IP) (Santa Cruz Biotechnology, SC-455); Rabbit antibodies against phospho-STAT5 Y694/699 (Cell Signaling Technology, 4322), phospho-AKT S473 (Cell Signaling Technology, 4060), p38 (Cell Signaling Technology, 8690), STAT5 (Cell Signaling Technology, 94205), AKT (Cell Signaling Technology, 4691), human IL-3Rα (for immunoblot) (MYBioSource, MBS2544022), murine Il-3rα (MYBioSource, MBS2542745), murine March2 (Abcam, ab220292), MARCH8 (Proteintech, 14119-1-AP), K48-linkage specific polyubiquitin (Abcam, ab140601); HRP-conjugated mouse anti-HA (Sigma-Aldrich, H6533); Goat anti-mouse IgG (Jackson Immuno Research, 115-035-003) were purchased from the indicated companies.

Techniques: Transfection, Immunoprecipitation, Western Blot, Sequencing