stealth sirna against rhbdl4 Search Results


rhbdl4  (Proteintech)
93
Proteintech rhbdl4
Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, <t>RHBDL4,</t> or SREBP1 using liver lysates of WT and A232E/+ mice. A , representative images of Western blot analyses for pSREBP1, pSREBP2, and β-actin protein levels in the plasma membrane fraction of the liver tissue. B and C , ratio of signal intensities of pSREBP1 ( B ) and pSREBP2 ( C ) to β-actin in Western blot analyses. D , representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E – G , ratio of signal intensities of nSREBP1 ( E ), nSREBP2 ( F ), and ChREBP ( G ) to TBP in Western blot analyses. H , immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I , immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J , immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B , C , E – G , values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.
Rhbdl4, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/stealth+sirna+against+rhbdl4/RHBDD1+Antibody/pmc09127370-263-36-38
Average 93 stars, based on 1 article reviews
rhbdl4 - by Bioz Stars, 2026-09
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mouse models for rhbdl4  (International Mouse Phenotyping Consortium)
86
International Mouse Phenotyping Consortium mouse models for rhbdl4
Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, <t>RHBDL4,</t> or SREBP1 using liver lysates of WT and A232E/+ mice. A , representative images of Western blot analyses for pSREBP1, pSREBP2, and β-actin protein levels in the plasma membrane fraction of the liver tissue. B and C , ratio of signal intensities of pSREBP1 ( B ) and pSREBP2 ( C ) to β-actin in Western blot analyses. D , representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E – G , ratio of signal intensities of nSREBP1 ( E ), nSREBP2 ( F ), and ChREBP ( G ) to TBP in Western blot analyses. H , immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I , immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J , immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B , C , E – G , values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.
Mouse Models For Rhbdl4, supplied by International Mouse Phenotyping Consortium, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/stealth+sirna+against+rhbdl4/for+models+mouse+rhbdl4/pmc12476590-73-8-31
Average 86 stars, based on 1 article reviews
mouse models for rhbdl4 - by Bioz Stars, 2026-09
86/100 stars
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gene exp rhbdl3 mm00460511 m1  (Thermo Fisher)
86
Thermo Fisher gene exp rhbdl3 mm00460511 m1
Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, <t>RHBDL4,</t> or SREBP1 using liver lysates of WT and A232E/+ mice. A , representative images of Western blot analyses for pSREBP1, pSREBP2, and β-actin protein levels in the plasma membrane fraction of the liver tissue. B and C , ratio of signal intensities of pSREBP1 ( B ) and pSREBP2 ( C ) to β-actin in Western blot analyses. D , representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E – G , ratio of signal intensities of nSREBP1 ( E ), nSREBP2 ( F ), and ChREBP ( G ) to TBP in Western blot analyses. H , immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I , immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J , immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B , C , E – G , values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.
Gene Exp Rhbdl3 Mm00460511 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/stealth+sirna+against+rhbdl4/Gene+Exp%2E+Rhbdl3%2C+Mm00460511_m1/pm17032746-122-28--1
Average 86 stars, based on 1 article reviews
gene exp rhbdl3 mm00460511 m1 - by Bioz Stars, 2026-09
86/100 stars
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rhbdl4 protein  (Gallus BioPharmaceuticals)
90
Gallus BioPharmaceuticals rhbdl4 protein
Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, <t>RHBDL4,</t> or SREBP1 using liver lysates of WT and A232E/+ mice. A , representative images of Western blot analyses for pSREBP1, pSREBP2, and β-actin protein levels in the plasma membrane fraction of the liver tissue. B and C , ratio of signal intensities of pSREBP1 ( B ) and pSREBP2 ( C ) to β-actin in Western blot analyses. D , representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E – G , ratio of signal intensities of nSREBP1 ( E ), nSREBP2 ( F ), and ChREBP ( G ) to TBP in Western blot analyses. H , immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I , immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J , immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B , C , E – G , values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.
Rhbdl4 Protein, supplied by Gallus BioPharmaceuticals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/stealth+sirna+against+rhbdl4/rhbdl4+protein/pmc10920636__41467_2024_45615_MOESM1_ESM-39-11-19
Average 90 stars, based on 1 article reviews
rhbdl4 protein - by Bioz Stars, 2026-09
90/100 stars
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w65a  (GenScript corporation)
90
GenScript corporation w65a
Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, <t>RHBDL4,</t> or SREBP1 using liver lysates of WT and A232E/+ mice. A , representative images of Western blot analyses for pSREBP1, pSREBP2, and β-actin protein levels in the plasma membrane fraction of the liver tissue. B and C , ratio of signal intensities of pSREBP1 ( B ) and pSREBP2 ( C ) to β-actin in Western blot analyses. D , representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E – G , ratio of signal intensities of nSREBP1 ( E ), nSREBP2 ( F ), and ChREBP ( G ) to TBP in Western blot analyses. H , immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I , immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J , immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B , C , E – G , values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.
W65a, supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/stealth+sirna+against+rhbdl4/w65a/pm39922490-234-1-11
Average 90 stars, based on 1 article reviews
w65a - by Bioz Stars, 2026-09
90/100 stars
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pcmv6 rhbdl4mycflag  (OriGene)
90
OriGene pcmv6 rhbdl4mycflag
Identification of PTP1B (also called PTPN1) in the BioID and as interactor of RHBDL4. A , peptide overage of PTP1B in the BioID of RHBDL4. Peptides identified by MS are shown in blue. B , the substrate-trapping mutant D181A and the super-trapping mutant D181A/Y46F of mCherry-PTP1B were transiently expressed with or without <t>RHBDL4mycFLAG.</t> The cells were lysed in immunoprecipitation ( IP )–compatible conditions and captured with anti-FLAG immunomagnetic beads. Co-immunoprecipitates and whole cell lysates ( Input ) were probed with anti-mCherry, or RHBDL4 antibody. C , HEK293 cells expressing TMEM115-mycFLAG or RHBDL4mycFLAG were lysed and bound with anti-FLAG beads ± 4 m m Na 3 VO 4 . The resulting co-immunoprecipitates were probed with anti-FLAG-HRP or anti-mCherry antibody.
Pcmv6 Rhbdl4mycflag, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/stealth+sirna+against+rhbdl4/RHBDD1+(NM_032276)+Human+Tagged+ORF+Clone/pmc06663880-210-14-19
Average 90 stars, based on 1 article reviews
pcmv6 rhbdl4mycflag - by Bioz Stars, 2026-09
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rhbdd1 nm 032276 human untagged clone  (OriGene)
N/A
RHBDD1 untagged Human rhomboid domain containing 1 RHBDD1 transcript variant 1
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rhbdl3 nm 139228 mouse tagged orf clone lentiviral particle  (OriGene)
N/A
Lenti ORF particles Rhbdl3 GFP tagged Mouse rhomboid veinlet like 3 Drosophila Rhbdl3 200ul 10 7 TU mL
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rhbdd1 nm 032276 human tagged orf clone  (OriGene)
N/A
RHBDD1 GFP tagged Human rhomboid domain containing 1 RHBDD1 transcript variant 1
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rhbdd1 nm 001167608 human tagged orf clone  (OriGene)
N/A
Lenti ORF clone of Human rhomboid domain containing 1 RHBDD1 transcript variant 2 Myc DDK tagged
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rhbdl3 nm 001105819 rat tagged orf clone  (OriGene)
N/A
Lenti ORF clone of Rhbdl3 mGFP tagged ORF Rat rhomboid veinlet like 3 Drosophila Rhbdl3 10 ug
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Image Search Results


Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, RHBDL4, or SREBP1 using liver lysates of WT and A232E/+ mice. A , representative images of Western blot analyses for pSREBP1, pSREBP2, and β-actin protein levels in the plasma membrane fraction of the liver tissue. B and C , ratio of signal intensities of pSREBP1 ( B ) and pSREBP2 ( C ) to β-actin in Western blot analyses. D , representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E – G , ratio of signal intensities of nSREBP1 ( E ), nSREBP2 ( F ), and ChREBP ( G ) to TBP in Western blot analyses. H , immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I , immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J , immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B , C , E – G , values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.

Journal: The Journal of Biological Chemistry

Article Title: AAA-ATPase valosin-containing protein binds the transcription factor SREBP1 and promotes its proteolytic activation by rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2022.101936

Figure Lengend Snippet: Protein expressions of precursor (p) and nuclear (n) forms of SREBP1 and co-immunoprecipitation assays for interactions of VCP with S1P, RHBDL4, or SREBP1 using liver lysates of WT and A232E/+ mice. A , representative images of Western blot analyses for pSREBP1, pSREBP2, and β-actin protein levels in the plasma membrane fraction of the liver tissue. B and C , ratio of signal intensities of pSREBP1 ( B ) and pSREBP2 ( C ) to β-actin in Western blot analyses. D , representative images of Western blot analyses for nSREBP1, nSREBP2, ChREBP, and TBP protein levels in the nuclear fraction of the liver tissue. E – G , ratio of signal intensities of nSREBP1 ( E ), nSREBP2 ( F ), and ChREBP ( G ) to TBP in Western blot analyses. H , immunoblots for S1P and VCP using proteins immunoprecipitated with anti-S1P antibody. I , immunoblots for RHBDL4 and VCP using proteins immunoprecipitated with anti-RHBDL4 antibody. J , immunoblots for SREBP1 and VCP using proteins immunoprecipitated with anti-SREBP1 antibody. Liver lysates of mice fed HFD were used for co-immunoprecipitation assays. Input indicates liver lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. B , C , E – G , values are means ± SEM (SD, n = 5 per group; HFD, n = 12–13 per group). Fold change is relative to WT mice fed SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ChREBP, carbohydrate-responsive element–binding protein; HFD, high-fat diet; SD, standard diet; S1P, site-1 protease; SREBP1, sterol regulatory element–binding protein 1; TBP, TATA box-binding protein; TG, triglyceride; VCP, valosin-containing protein.

Article Snippet: Antibodies used in the present study are as follows: SREBP1 (ab3259; Abcam), VCP (ab11433; Abcam), S1P (ab140592; Abcam), SREBP2 (ab30682; Abcam), CHOP (2895; Cell Signaling Technology), BiP (3177; Cell Signaling Technology), β-actin (3700; Cell Signaling Technology), RHBDL4 (20869-1-AP; Proteintech), TBP (22006-1-AP; Proteintech), gp78 (16675-1-AP; Proteintech), HRD1 (13473-1-AP; Proteintech), Ubiquitin (10201-2-AP; Proteintech), carbohydrate-responsive element–binding protein (NB400-135; Novus Biologicals), ATF6 (NBP1-40256; Novus Biologicals), and normal mouse IgG (sc-2025; Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Western Blot, Clinical Proteomics, Membrane, Control, Binding Assay

Knockdown experiments of VCP or RHBDL4 with siRNA and inhibition experiment of VCP with an allosteric inhibitor in HepG2 cells. A – C , Western blot analyses for VCP, pSREBP1, nSREBP1, full-length ATF6, cleaved ATF6, and β-actin protein levels in HepG2 cells with or without VCP siRNA ( A ), NMS-873, an allosteric VCP inhibitor, ( B ) or RHBDL4 siRNA ( C ). D – F , mRNA expressions of FAS and SCD1 in HepG2 cells with or without VCP siRNA ( D ), NMS-873 ( E ), or RHBDL4 siRNA ( F ) (n = 3). mRNA expression levels were normalized by GAPDH. The fold change is relative to HepG2 cells transfected with control siRNA ( D and F ) or HepG2 cells treated with DMSO ( E ). Values are means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ATF6, activating transcription factor 6; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1; SREBP1, sterol regulatory element-binding protein 1; VCP, valosin-containing protein.

Journal: The Journal of Biological Chemistry

Article Title: AAA-ATPase valosin-containing protein binds the transcription factor SREBP1 and promotes its proteolytic activation by rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2022.101936

Figure Lengend Snippet: Knockdown experiments of VCP or RHBDL4 with siRNA and inhibition experiment of VCP with an allosteric inhibitor in HepG2 cells. A – C , Western blot analyses for VCP, pSREBP1, nSREBP1, full-length ATF6, cleaved ATF6, and β-actin protein levels in HepG2 cells with or without VCP siRNA ( A ), NMS-873, an allosteric VCP inhibitor, ( B ) or RHBDL4 siRNA ( C ). D – F , mRNA expressions of FAS and SCD1 in HepG2 cells with or without VCP siRNA ( D ), NMS-873 ( E ), or RHBDL4 siRNA ( F ) (n = 3). mRNA expression levels were normalized by GAPDH. The fold change is relative to HepG2 cells transfected with control siRNA ( D and F ) or HepG2 cells treated with DMSO ( E ). Values are means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. ATF6, activating transcription factor 6; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1; SREBP1, sterol regulatory element-binding protein 1; VCP, valosin-containing protein.

Article Snippet: Antibodies used in the present study are as follows: SREBP1 (ab3259; Abcam), VCP (ab11433; Abcam), S1P (ab140592; Abcam), SREBP2 (ab30682; Abcam), CHOP (2895; Cell Signaling Technology), BiP (3177; Cell Signaling Technology), β-actin (3700; Cell Signaling Technology), RHBDL4 (20869-1-AP; Proteintech), TBP (22006-1-AP; Proteintech), gp78 (16675-1-AP; Proteintech), HRD1 (13473-1-AP; Proteintech), Ubiquitin (10201-2-AP; Proteintech), carbohydrate-responsive element–binding protein (NB400-135; Novus Biologicals), ATF6 (NBP1-40256; Novus Biologicals), and normal mouse IgG (sc-2025; Santa Cruz Biotechnology).

Techniques: Knockdown, Inhibition, Western Blot, Expressing, Transfection, Control, Binding Assay

Knockdown experiments of E3 ligase, gp78, or HRD1 with siRNA in HepG2 cells. A and B , Western blot analyses for gp78, pSREBP1, nSREBP1, and β-actin protein levels with or without gp78 siRNA ( A ) and for HRD1, pSREBP1, nSREBP1, and β-actin protein levels with or without HRD1 siRNA ( B ). C and D , mRNA expressions of FAS and SCD1 with or without gp78 siRNA ( C ) and with or without HRD1 siRNA ( D ) (n = 3). mRNA expression levels were normalized by GAPDH. The fold change is relative to HepG2 cells transfected with control siRNA. E and F , Western blot analyses for SREBP1, gp78, VCP, RHBDL4, and ubiquitin using proteins immunoprecipitated with anti-SREBP1 antibody with or without gp78 siRNA ( E ) and for SREBP1, HRD1, VCP, RHBDL4, and ubiquitin using proteins immunoprecipitated with anti-SREBP1 antibody with or without HRD1 siRNA ( F ). Input indicates cell lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. C and D , values are means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. FAS, fatty acid synthase; HRD1, HMG-CoA reductase degradation protein 1; SCD1, stearoyl-CoA desaturase 1; SREBP1, sterol regulatory element-binding protein 1; VCP, valosin-containing protein.

Journal: The Journal of Biological Chemistry

Article Title: AAA-ATPase valosin-containing protein binds the transcription factor SREBP1 and promotes its proteolytic activation by rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2022.101936

Figure Lengend Snippet: Knockdown experiments of E3 ligase, gp78, or HRD1 with siRNA in HepG2 cells. A and B , Western blot analyses for gp78, pSREBP1, nSREBP1, and β-actin protein levels with or without gp78 siRNA ( A ) and for HRD1, pSREBP1, nSREBP1, and β-actin protein levels with or without HRD1 siRNA ( B ). C and D , mRNA expressions of FAS and SCD1 with or without gp78 siRNA ( C ) and with or without HRD1 siRNA ( D ) (n = 3). mRNA expression levels were normalized by GAPDH. The fold change is relative to HepG2 cells transfected with control siRNA. E and F , Western blot analyses for SREBP1, gp78, VCP, RHBDL4, and ubiquitin using proteins immunoprecipitated with anti-SREBP1 antibody with or without gp78 siRNA ( E ) and for SREBP1, HRD1, VCP, RHBDL4, and ubiquitin using proteins immunoprecipitated with anti-SREBP1 antibody with or without HRD1 siRNA ( F ). Input indicates cell lysates without immunoprecipitation. IgG indicates control proteins immunoprecipitated with isotype antibody. Spliced-together lanes were run on the same gel but were noncontiguous. C and D , values are means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005 as determined by 2-tailed Student’s t test for comparing two groups. FAS, fatty acid synthase; HRD1, HMG-CoA reductase degradation protein 1; SCD1, stearoyl-CoA desaturase 1; SREBP1, sterol regulatory element-binding protein 1; VCP, valosin-containing protein.

Article Snippet: Antibodies used in the present study are as follows: SREBP1 (ab3259; Abcam), VCP (ab11433; Abcam), S1P (ab140592; Abcam), SREBP2 (ab30682; Abcam), CHOP (2895; Cell Signaling Technology), BiP (3177; Cell Signaling Technology), β-actin (3700; Cell Signaling Technology), RHBDL4 (20869-1-AP; Proteintech), TBP (22006-1-AP; Proteintech), gp78 (16675-1-AP; Proteintech), HRD1 (13473-1-AP; Proteintech), Ubiquitin (10201-2-AP; Proteintech), carbohydrate-responsive element–binding protein (NB400-135; Novus Biologicals), ATF6 (NBP1-40256; Novus Biologicals), and normal mouse IgG (sc-2025; Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Expressing, Transfection, Control, Ubiquitin Proteomics, Immunoprecipitation, Binding Assay

Identification of PTP1B (also called PTPN1) in the BioID and as interactor of RHBDL4. A , peptide overage of PTP1B in the BioID of RHBDL4. Peptides identified by MS are shown in blue. B , the substrate-trapping mutant D181A and the super-trapping mutant D181A/Y46F of mCherry-PTP1B were transiently expressed with or without RHBDL4mycFLAG. The cells were lysed in immunoprecipitation ( IP )–compatible conditions and captured with anti-FLAG immunomagnetic beads. Co-immunoprecipitates and whole cell lysates ( Input ) were probed with anti-mCherry, or RHBDL4 antibody. C , HEK293 cells expressing TMEM115-mycFLAG or RHBDL4mycFLAG were lysed and bound with anti-FLAG beads ± 4 m m Na 3 VO 4 . The resulting co-immunoprecipitates were probed with anti-FLAG-HRP or anti-mCherry antibody.

Journal: The Journal of Biological Chemistry

Article Title: Spatial proteomics reveal that the protein phosphatase PTP1B interacts with and may modify tyrosine phosphorylation of the rhomboid protease RHBDL4

doi: 10.1074/jbc.RA118.007074

Figure Lengend Snippet: Identification of PTP1B (also called PTPN1) in the BioID and as interactor of RHBDL4. A , peptide overage of PTP1B in the BioID of RHBDL4. Peptides identified by MS are shown in blue. B , the substrate-trapping mutant D181A and the super-trapping mutant D181A/Y46F of mCherry-PTP1B were transiently expressed with or without RHBDL4mycFLAG. The cells were lysed in immunoprecipitation ( IP )–compatible conditions and captured with anti-FLAG immunomagnetic beads. Co-immunoprecipitates and whole cell lysates ( Input ) were probed with anti-mCherry, or RHBDL4 antibody. C , HEK293 cells expressing TMEM115-mycFLAG or RHBDL4mycFLAG were lysed and bound with anti-FLAG beads ± 4 m m Na 3 VO 4 . The resulting co-immunoprecipitates were probed with anti-FLAG-HRP or anti-mCherry antibody.

Article Snippet: All point mutations were introduced by site-directed mutagenesis using CloneAmp HiFI PCR premix (Clontech). pCMV6 RHBDL4mycFLAG was purchased from Origene (RC210708).

Techniques: Mutagenesis, Immunoprecipitation, Expressing

Co-immunoprecipitation ( IP ) of Lys 63 -linked ubiquitin and p97/VCP with mutants of RHBDL4. HEK293 cells expressing myc-FLAG–tagged mutants of RHBDL4 for site 1 (Y206F), site 7 (Y261F), site 8 (Y264F), and all-sites mutant were lysed and incubated with anti-FLAG beads. Co-immunoprecipitates and whole cell lysates ( Input ) were probed with anti-p97/VCP ( panel a ), anti-ubiquitin ( panel b ), anti-Lys 63 linkage–specific antibodies ( panel c ), and anti-FLAG-HRP for RHBDL4mycFLAG ( panel d ). B , relative quantification of the immunoreactivity of co-immunoprecipitates ( panel a ) versus anti-p97/VCP (*, p = 0.014, n = 3) ( panel b ) or anti-Lys 63 -linked ubiquitin (*, p = 0.024, n = 3) of WT RHBDL4mycFLAG and AllY→F, normalized against the input. The error bars represent standard deviation from the means.

Journal: The Journal of Biological Chemistry

Article Title: Spatial proteomics reveal that the protein phosphatase PTP1B interacts with and may modify tyrosine phosphorylation of the rhomboid protease RHBDL4

doi: 10.1074/jbc.RA118.007074

Figure Lengend Snippet: Co-immunoprecipitation ( IP ) of Lys 63 -linked ubiquitin and p97/VCP with mutants of RHBDL4. HEK293 cells expressing myc-FLAG–tagged mutants of RHBDL4 for site 1 (Y206F), site 7 (Y261F), site 8 (Y264F), and all-sites mutant were lysed and incubated with anti-FLAG beads. Co-immunoprecipitates and whole cell lysates ( Input ) were probed with anti-p97/VCP ( panel a ), anti-ubiquitin ( panel b ), anti-Lys 63 linkage–specific antibodies ( panel c ), and anti-FLAG-HRP for RHBDL4mycFLAG ( panel d ). B , relative quantification of the immunoreactivity of co-immunoprecipitates ( panel a ) versus anti-p97/VCP (*, p = 0.014, n = 3) ( panel b ) or anti-Lys 63 -linked ubiquitin (*, p = 0.024, n = 3) of WT RHBDL4mycFLAG and AllY→F, normalized against the input. The error bars represent standard deviation from the means.

Article Snippet: All point mutations were introduced by site-directed mutagenesis using CloneAmp HiFI PCR premix (Clontech). pCMV6 RHBDL4mycFLAG was purchased from Origene (RC210708).

Techniques: Immunoprecipitation, Ubiquitin Proteomics, Expressing, Mutagenesis, Incubation, Quantitative Proteomics, Standard Deviation