Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: A Chaperone Complex Formed by HSP47, FKBP65 and BiP Modulates Telopeptide Lysyl Hydroxylation of Type I Procollagen

doi: 10.1002/jbmr.3095

Figure Lengend Snippet: LH2, FKBP65, HSP47 and BiP interact. A-C. Protein levels of LH2 were altered in cells with mutations in FKBP10 and SERPINH1. BiP levels were decreased in HSP47 defective cells. D-E. Rescue experiment using FKBP10-/- cells transfected with FKBP10 tagged vector. LH2 levels were rescued by expression of wildtype FKBP65 protein. F. Co-immunoprecipitation of endogenous LH2, FKBP65, HSP47 and BiP in human osteoblasts (OB) with specific antibodies. LH2 immunoprecipitated HSP47 and BiP. G. Co-immunoprecipitation of tagged FKBP65 and LH2 in OB cells. LH2 and FKBP65 immunoprecipitated each other, including both LH2 isoforms, respectively.

Article Snippet: For co-immunoprecipitation of tagged proteins, DDK fusion vectors and an anti-DDK Magnetic Immunoprecipitation and detection kit ( {"type":"entrez-nucleotide","attrs":{"text":"AR100024","term_id":"12810472","term_text":"AR100024"}} AR100024 Origene) were used.

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: A Chaperone Complex Formed by HSP47, FKBP65 and BiP Modulates Telopeptide Lysyl Hydroxylation of Type I Procollagen

doi: 10.1002/jbmr.3095

Figure Lengend Snippet: Proteins identified by Immunoprecipitation followed by Mass Spectrometry

Article Snippet: For co-immunoprecipitation of tagged proteins, DDK fusion vectors and an anti-DDK Magnetic Immunoprecipitation and detection kit ( {"type":"entrez-nucleotide","attrs":{"text":"AR100024","term_id":"12810472","term_text":"AR100024"}} AR100024 Origene) were used.

Techniques: Immunoprecipitation, Mass Spectrometry

Journal: Cell reports

Article Title: Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5

doi: 10.1016/j.celrep.2023.113145

Figure Lengend Snippet:

Article Snippet: MYC Epitope Tag Antibody Rabbit Polyclonal , Rockland , Cat#600-401-381; RRID:AB_217927.

Techniques: Control, Recombinant, Affinity Purification, Virus, Plasmid Preparation, Quantitative Proteomics, Over Expression, Knock-Out, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: RREB-1 is a transcriptional repressor of HLA-G.

doi: 10.4049/jimmunol.0902053

Figure Lengend Snippet: FIGURE 8. In situ binding of RREB-1 or HDAC1 to the HLA-G pro- moter in a repressive and active-type chromatin. A and B, ChIP performed with JEG-3 (HLA-G ) and M8 (HLA-G ) cells using anti-RREB-1 and anti-HDAC1 Abs on distal and proximal promoter regions (A) Abs target- ing RNA polymerase II (RNApolII), acetylated histone H3 (AcH3), and phosphorylated histone H3 (AcH3 P) on proximal promoter region (B). Immunoprecipitated HLA-G promoter regions are analyzed on agarose gels by semiquantitative HLA-G-specific PCRs targeting proximal and distal HLA-G promoter. Input chromatin (Input) used as PCR control and IgG () are shown. The absence of RREB-1 and HDAC1 binding observed in JEG-3 cells and the absence of RNA polymerase II, acetylated histone H3, and phosphorylated histone H3 binding in M8 cells validate the specificity of Abs used in ChIP assays.

Article Snippet: ChIP assays were performed as previously described (51) using antiRREB-1 from Rockland; anti-acetylated histone H3 (06-599) and antiphosphorylated Ser10 histone H3 (07-081) from Upstate Biotechnology Associates; and anti-RNApolII (C-21) and anti-HDAC1 (H-51) from Santa Cruz Biotechnology.

Techniques: In Situ, Binding Assay, Immunoprecipitation, Control

Journal: Nature chemical biology

Article Title: A chemoproteomic portrait of the oncometabolite fumarate

doi: 10.1038/s41589-018-0217-y

Figure Lengend Snippet: Functional analyses of FH -regulated Cys residues. (a) Percentage of FH -regulated Cys residues predicted to be functional using the informatics tool Mutation Assessor (R ≥1.5, n ≥2). (b) Gene ontology analysis of FH -regulated Cys residues. (c) Correlation between genes containing FH -regulated Cys residues and genetic alterations in kidney cancer (RCC). Statistical significance was assessed using Student’s t -test (two-tailed, unpaired); ** P < 0.01. (d) SMARCC1 C520 lies in the SWIRM domain and is highly conserved. (e) SMARCC1 C520 lies at the SNF5 subunit interface. (f) SMARCC1 C520 undergoes FH -regulated changes in occupancy in HLRCC cells ( n = 3 independent experiments). (g) SMARCC1 C520E mutation limits co-immunoprecipitation with SNF5 in HEK-293 cells co-overexpressing FLAG-tagged SNF5 with Myc-tagged SMARCC1. (h) SMARCC1 S-succination can be detected in FH-deficient and FH WT HLRCC cell lines after IP of endogenous SMARCC1. (i) SNF5 demonstrates decreased co-immunoprecipitation and decreased levels in FH−/− HLRCC cells. Left: Results from SWI/SNF complex co-immunoprecipitation with BRG1 antibody. Right: Endogenous levels of SWI/SNF complex members in HLRCC cells. Representative images from two independent experiments are shown in g-i. Uncropped scans of immunoblots are provided in . (j) EZH2 inhibitors exhibit modest selectivity for FH-deficient HLRCC cells. UOK262 FH −/− or FH WT spheroids were treated with GSK126 (21 days; 1, 2, 4, 8, 16 μM) and % viability plotted relative to the vehicle treated spheroids. Data is presented as mean ± s.d.; n = 3/group. (k) EZH2 inhibitors are toxic to HLRCC spheroids. UOK262 FH −/− spheroids were treated with vehicle or EPZ6438 (14 days; 5 μM). Figure is representative of 6 replicates.

Article Snippet: Anti-FLAG pulldown was performed using immunoprecipitation kit (KBA-319–383) from Rockland Immunochemicals, Inc. SDS-PAGE was performed using Bis-Tris NuPAGE gels (4–12%, Invitrogen #NP0322), and MES running buffer (Life technologies #NP0002) in Xcell SureLock MiniCells (Invitrogen) according to the manufacturer’s instructions.

Techniques: Functional Assay, Mutagenesis, Two Tailed Test, Immunoprecipitation, Western Blot