Recombinant Human SMPDL3B protein, His-tagged

• Cat.No.: SMPDL3B-2822H
• Product Overview: Recombinant Human SMPDL3B protein(1-336 aa), fused with N-terminal His tag, was expressed in E.coli.

Specification

• Species: Human
• Source: E.coli
• Tag: His
• Protein Length: 1-336 aa
• Form: The purified protein was lyophilized from sterile phosphate-buffered saline (PBS: 58 mM Na₂HPO₄, 17 mM NaH₂PO₄, 68 mM NaCl, pH 7.4). Prior to lyophilization, 5% (w/v) trehalose and 5% (w/v) mannitol were incorporated as cryoprotective excipients. The protein was eluted with a buffer containing 300 mM imidazole.
• AASequence: MRLLAWLIFLANWGGARAEPGKFWHIADLHLDPDYKVSKDPFQVCPSAGSQPVPDAGPWGDYLCDSPWALINSSIYAMKEIEPEPDFILWTGDDTPHVPDEKLGEAAVLEIVERLTKLIREVFPDTKVYAALGNHDFHPKNQFPAGSNNIYNQIAELWKPWLSNESIALFKKGAFYCEKLPGPSGAGRIVVLNTNLYYTSNALTADMADPGQQFQWLEDVLTDASKAGDMVYIVGHVPPGFFEKTQNKAWFREGFNEKYLKVVRKHHRVIAGQFFGHHHTDSFRMLYDDAGVPISAMFITPGVTPWKTTLPGVVNGANNPAIRVFEYDRATLSLKV
• Purity: 85%, by SDS-PAGE with Coomassie Brilliant Blue staining.
• Storage: Store at 2-8°C for 1-2 weeks. Aliquot and store at -20°C to -80°C for up to 3 months, reconstitution with sterile water and addition of an equal volume of glycerol. Avoid repeat freeze-thaw cycles.
• Reconstitution: Dissolve the product in 200 μl sterile water to achieve a working concentration of 0.25 µg/μl. If experimental compatibility allows, mix the aqueous solution with an equal volume of glycerol (1:1 ratio) after initial reconstitution.

Gene Information

• Gene Name: SMPDL3B sphingomyelin phosphodiesterase, acid-like 3B [ Homo sapiens ]
• Official Symbol: SMPDL3B
• Synonyms: SMPDL3B; sphingomyelin phosphodiesterase, acid-like 3B; acid sphingomyelinase-like phosphodiesterase 3b; ASML3B; ASM-like phosphodiesterase 3b
• Gene ID: 27293
• mRNA Refseq: NM_001009568
• Protein Refseq: NP_001009568
• UniProt ID: Q92485

Citation

SMPDL3b modulates insulin receptor signaling in diabetic kidney disease

Journal: Nature Communications    PubMed ID: 31217420    Data: 2019/6/19

Authors: A. Mitrofanova, S. K. Mallela, A. Fornoni

Article Snippet:C16:0 C1P (#860533) and C6-NBD-Ceramide (#810209P) were obtained from Avanti Polar Lipid (AL, USA).C16:0 C1P (#860533) and C6-NBD-Ceramide (#810209P) were obtained from Avanti Polar Lipid (AL, USA).. Recombinant human SMPDL3b protein, MYC/DDK-tagged was ordered from Creative BioMart (#SMPDL3b-2823H).. Anti-caveolin-1 (#3251 for phospho-Cav1(Tyr14), dilution 1:1000, #3267 for Cav1 (D46G3) XP TM , dilution 1:1000), anti-IR β subunit (4B8) (#3025, dilution 1:100), anti-Na/K-ATPase (#3010, dilution 1:1000), anti-MEK-1/2 (D1A5) (#8727, dilution 1:1,000), anti-AKT (#9271 for phospho-AKT (Ser473) (D9E), dilution 1:1000, #9272 for tAKT, dilution 1:1000), anti-p70S6Ke (#9234 for phosphor-p70S6K (Thr389 (108D2)), dilution 1:1000, #9202 for p70S6K, dilution 1:1000), anti-4EBP1 (#2855 for phospho-4E-BP1 (Thr37/46) (236B4), dilution 1:1000, #9644 for 4E-BP1, dilution 1:1000) primary antibodies were purchased from Cell Signaling Technology (MA, USA).Anti-caveolin-1 (#3251 for phospho-Cav1(Tyr14), dilution 1:1000, #3267 for Cav1 (D46G3) XP TM , dilution 1:1000), anti-IR β subunit (4B8) (#3025, dilution 1:100), anti-Na/K-ATPase (#3010, dilution 1:1000), anti-MEK-1/2 (D1A5) (#8727, dilution 1:1,000), anti-AKT (#9271 for phospho-AKT (Ser473) (D9E), dilution 1:1000, #9272 for tAKT, dilution 1:1000), anti-p70S6Ke (#9234 for phosphor-p70S6K (Thr389 (108D2)), dilution 1:1000, #9202 for p70S6K, dilution 1:1000), anti-4EBP1 (#2855 for..

SMPDL3b affects the conversion of C1P to ceramide. a Bar graph analysis of phosphodiesterase (PDE) activity in control (CTRL), SMPDL3b knockdown (siSMP) and SMPDL3b overexpressing (SMP OE) human podocytes. n = 3 independent experiments; * P = 0.039, two-tailed t -test. b Liquid chromatography mass spectrometry analysis (LC–MS) of total sphingomyelin content in CTRL, siSMP, and SMP OE human podocytes. n = 3 independent experiments. P = 0.78, F = 0.26, one-way ANOVA. c LC–MS of total ceramide content in CTRL, siSMP, and SMP OE human podocytes. n = 3 independent experiments. P = 0.78, F = 0.78, one-way ANOVA. d LC–MS of total ceramide-1-phosphate (C1P) content in CTRL, siSMP, and SMP OE human podocytes. n = 3 independent experiments. * P = 0.045, ** P = 0.007, F = 32.58, one-way ANOVA. e C1P phosphatase in vitro assay using increasing amounts C6-NBD-C1P. n = 2 independent experiments in duplicate; * P < 0.05, *** P < 0.001; two-tailed t -test. Error bars represent standard deviation (SD)

SMPDL3b regulates the insulin-signaling pathway in human podocytes. a Representative Western blot image of main downstream targets of the insulin receptor signaling in control (CTRL), SMPDL3b knockdown (siSMP) and SMPDL3b overexpressing (SMP OE) podocytes exposed to increasing concentration of insulin (0, 0.1, 1 nM): phosphorylated (pAKT) and total (tAKT) AKT; phosphorylated (p-p70S6K) and total (t-p70S6K) p70S6K; phosphorylated (p-4EB-P1) and total (t-4EB-P1) 4EB-P1. b Bar graph analysis of the fold change ratio of pAKT to tAKT (left panel), p-p70S6K to t-p70S6K (central panel) and p-4EB-P1 to t-4EB-P1 (right panel) in CTRL, siSMP, and SMP OE podocytes. n = 3 independent experiments; * P < 0.05 (when compared AKT phosphorylation level in insulin treated CTRL cells); ## P = 0.002, # P = 0.011, F = 5.67 (when compared AKT phosphorylation level in insulin-treated siSMP cells); *** P = 0.0002, **** P < 0.0001, F = 21.47 (when compared p70S6K phosphorylation level in insulin-treated SMP OE cells); $$ P = 0.008, $$$$ P < 0.0001, F = 10.09 (when compared 4EB-P1 phosphorylation level in insulin-treated SMP OE cells); one-way ANOVA. n = 3 independent experiments. c Kinetics of AKT (left panel), p70S6K (central panel), and 4EB-P1 (right panel) phosphorylation in CTRL, siSMP, and SMP OE podocytes treated with 1 nM of insulin. ** P = 0.001, F = 9.31; ## P = 0.0003, F = 12.27; $$ P = 0.003, F = 7.84, one-way ANOVA. n = 3. d Western blot and bar graph analysis of insulin receptor (IR) expression in total lysates of CTRL and SMP OE podocytes; P = 0.448, two-tailed t -test. n = 4 independent experiments. e Western blot and graph analysis of IR localization at the plasma membrane (PM) in CTRL and SMP OE podocytes. Na/K-ATPase was used as a marker for the PM fraction and MEK-1/2 was used as a marker of the cytosolic fraction (CYTO); *** P = 0.0004, two-tailed t -test. n = 5 independent experiments. f FACS and related quantification analysis of the insulin receptor expression in CTRL and SMP OE podocytes. Unstained cells were used as a negative control; HEK293 cells transfected with an insulin receptor overexpressing construct were served as a positive control. A total of 30,000 cells per sample was analyzed; *** P < 0.0001, two-tailed t -test. n = 4 independent experiments. g PCR analysis of the insulin receptor isoform A (IRA) and isoform B (IRB) gene expression in CTRL, and SMP OE podocytes. Error bars represent standard deviation (SD)

SMPDL3b interacts with IRA, IRB, and caveolin-1 in a competitive manner. a – c Immunoprecipitation (IP) experiments performed in HEK293 cells co-transfected with human hIRA-FLAG, hIRB-FLAG, hSMPDL3b-GFP, hSMPDL3b-HA hSMPDL3b-H135A-HA, hCav1-FLAG, and hCav1-GFP plasmids. Empty FLAG vector (Empty-FLAG) was used as a negative control. Antibodies (IB) against FLAG, SMPDL3b, insulin receptor β-subunit (IR), or caveolin-1 (Cav1) were used in Western blot analysis. Each IP experiment was repeated at least three times. a SMPDL3b interacts with both IR isoforms but preferentially with IRA. b Wildtype SMPDL3b interacts with IRA, IRB, and caveolin-1, a mutation (H135A) in the phosphodiesterase activity domain of SMPDL3b does not affect these interactions. c Overexpression of SMPDL3b enhances the interaction between caveolin-1 and IRA (left panel) and suppresses the interaction between caveolin-1 and IRB (right panel). d Endogenous IP experiments using glomeruli isolated from five C57BL/6 mice indicate that SMPDL3b immunoprecipitates insulin receptor and caveolin-1. IgG served as a negative control. Each IP was repeated three times. e Western blot and bar graph analysis of AKT and p70S6K phosphorylation in control (CTRL) and SMPDL3b overexpressing (SMP OE) podocytes pre-treated with 5 mM methyl-β-cyclodextrin (CD) and stimulated with 1 nM insulin; * P = 0.02, ** P = 0.001, two-tailed t -test. n = 3 independent experiments. f IP experiments performed in HEK293 cells co-transfected with human hIRA-FLAG, hIRB-FLAG, hCav1-GFP, and hSMPDL3b-HA show that 5 mM CD treatment abrogates IRA/Cav1 interaction and restores IRB/Cav1 interaction in presence of SMPDL3b overexpression. g Western blot and bar graph analysis of the insulin receptor (IR) and SMPDL3b localization at the plasma membrane (PM) in CTRL and SMP OE podocytes exposed to 5 mM CD for 1 h; * P = 0.049, *** P = 0.0001 (for IR); $ P = 0.024, # P = 0.031 (for SMPDL3b); two-tailed t -test. n = 3 independent experiments. Error bars represent standard deviation (SD)