Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

doi: 10.1073/pnas.1600074113

Figure Lengend Snippet: LH2 splice variants, but not LH1 and LH3, interact with FKBP65. (A) Western blot detection of 3×FLAG-tag co-IP complexes from HEK293T cells overexpressed with combinations of LH2A-FLAG, LH2B-FLAG, and FKBP65. (B) Western blot detection of 6×His-tag co-IP complexes from HEK293T cells overexpressed with combinations of LH1-His, LH2-His, and FKBP65.

Article Snippet: FKBP65, LH2A, and LH2B cDNA ORF coding plasmids (Origene) were subcloned either directly into a pcDNA3.1 vector with primers introducing 6×His-tag to their C terminus or via a pCDNA3.1 ligated linker region to obtain in-frame 3×FLAG to the C terminus ( ).

Techniques: Western Blot, Co-Immunoprecipitation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

doi: 10.1073/pnas.1600074113

Figure Lengend Snippet: Catalytic inactive LH2 and Bruck syndrome mutated FKBP65 do not negatively affect complex formation. (A) Illustration of the functional domains of LH2A, LH2B, and FKBP65 (UniProt). Locations of the introduced point mutations are indicated in red. LH2 hydroxylase activity was blocked by mutations (red) in the Fe2-OG dioxygenase domain (green), whereas two known Bruck syndrome (BS) mutations were introduced in the FKBP65 PPIase domain 1. (B) Western blot detection of 3×FLAG-tag co-IP complexes from HEK293T cells overexpressed with combinations of catalytic inactive mutants of LH2A-FLAG and LH2B-FLAG with FKBP65. (C) Western blot detection of 3×FLAG-tag co-IP complexes from HEK293T cells overexpressed with combinations LH2A-FLAG, LH2B-FLAG with 6×His-tag WT (FKBP65-His), or BS mutated FKBP65 (FKBP65ΔΔ-His).

Article Snippet: FKBP65, LH2A, and LH2B cDNA ORF coding plasmids (Origene) were subcloned either directly into a pcDNA3.1 vector with primers introducing 6×His-tag to their C terminus or via a pCDNA3.1 ligated linker region to obtain in-frame 3×FLAG to the C terminus ( ).

Techniques: Functional Assay, Activity Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

doi: 10.1073/pnas.1600074113

Figure Lengend Snippet: LH2 splice variants can form heterodimers with LH1 and LH3. (A) Western blot detection of 3×FLAG-tag coimmunoprecipitated heterodimer complexes from HEK293T cells overexpressed with combinations of LH1-His and LH3-His with LH2A-FLAG. (B) Western blot detection of 3×FLAG-tag coimmunoprecipitated heterodimer complexes from HEK293T cells overexpressed with combinations of LH1-His and LH3-His with LH2B-FLAG. (C) Western blot detection of 3×FLAG-tag coimmunoprecipitated heterodimer complexes from HEK293T cells overexpressed with combinations of LH2A-FLAG and LH2B-His.

Article Snippet: FKBP65, LH2A, and LH2B cDNA ORF coding plasmids (Origene) were subcloned either directly into a pcDNA3.1 vector with primers introducing 6×His-tag to their C terminus or via a pCDNA3.1 ligated linker region to obtain in-frame 3×FLAG to the C terminus ( ).

Techniques: Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

doi: 10.1073/pnas.1600074113

Figure Lengend Snippet: Oligos used to generate affinity tags and site-directed mutagenesis by PCR

Article Snippet: FKBP65, LH2A, and LH2B cDNA ORF coding plasmids (Origene) were subcloned either directly into a pcDNA3.1 vector with primers introducing 6×His-tag to their C terminus or via a pCDNA3.1 ligated linker region to obtain in-frame 3×FLAG to the C terminus ( ).

Techniques: Mutagenesis

Journal: Cancer Medicine

Article Title: Gene expression of the IGF pathway family distinguishes subsets of gastrointestinal stromal tumors wild type for KIT and PDGFRA

doi: 10.1002/cam4.57

Figure Lengend Snippet: Target gene expression relative to GAPDH in GIST subtypes

Article Snippet: Commercial plasmids (OriGene, Rockville, MD) included clones of IGF1 (RG215257), IGF2 (RC201849), INSR (RG215257), IRS1 (SC124032), CDH2 (SC119018), NEFL (SC116280), LHX2 (SC126761), KIRREL3 (SC100705), CD34 (SC302353), and HIF1A (SC119189).

Techniques: Expressing

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Development of a High Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small Molecule Inhibitors Against LH2

doi: 10.1177/2472555218817057

Figure Lengend Snippet: Assays were performed by varying A) LH2 concentrations (0 – 1 μM), B) [IKG]3 peptide concentrations (0–1000 μM), and C) time (5, 60, 120 or 180 min; with (●) and without (■) LH2). D) A succinate standard curve fitted linearly (R2 = 0.9988).

Article Snippet: Peptide substrates for LH2 (IKGIKGIKG, abbreviated as [IKG] 3 ) and L230 (GTKGETGLKGII, abbreviated as GI-12) were ordered from LifeTein (Somerset, NJ) and the purity was determined to be greater than 98% by HPLC.

Techniques:

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Development of a High Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small Molecule Inhibitors Against LH2

doi: 10.1177/2472555218817057

Figure Lengend Snippet: Activity of LH2 (in %) were measured against variations of A) BSA, B) DMSO, C) triton X-100, D) tween-20, E) NP-40, and F) incubation time. In all cases, a positive control with 0% variation is normalized to 100 % activity. % activity of LH2 assays containing variations were calculated according to [xix0∗100], where x0 is RLU for positive control and xi is RLU for varying samples.

Article Snippet: Peptide substrates for LH2 (IKGIKGIKG, abbreviated as [IKG] 3 ) and L230 (GTKGETGLKGII, abbreviated as GI-12) were ordered from LifeTein (Somerset, NJ) and the purity was determined to be greater than 98% by HPLC.

Techniques: Activity Assay, Incubation, Positive Control

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Development of a High Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small Molecule Inhibitors Against LH2

doi: 10.1177/2472555218817057

Figure Lengend Snippet: A) Scattered plot of z’ values for all assay plates. B) Histogram showing percent inhibition of LH2 activity against frequency of compounds (red, peak 1, 0% inhibition control; blue, peak 2, 100% inhibition control; green, peak 3, % inhibition by library compounds). C) Flowchart of hits identification and confirmation process.

Article Snippet: Peptide substrates for LH2 (IKGIKGIKG, abbreviated as [IKG] 3 ) and L230 (GTKGETGLKGII, abbreviated as GI-12) were ordered from LifeTein (Somerset, NJ) and the purity was determined to be greater than 98% by HPLC.

Techniques: Inhibition, Activity Assay, Control

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Development of a High Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small Molecule Inhibitors Against LH2

doi: 10.1177/2472555218817057

Figure Lengend Snippet: X-axis represents different concentrations of compounds (in log[M]) whereas Y-axis represents inhibition of LH2 activity (in percent). In all cases, a no compound (0% inhibition) control was included and used as reference to calculate percent inhibition values. Percent inhibition was calculated according to equation (2) and data were fitted to four parameter logistic non-linear regression. Compounds 1 (●), 2 (■) and 3 (◆) show pIC50 values of 6.94 ± 0.08, 6.52 ± 0.04 and 6.32 ± 0.10, respectively, where pIC50 = −log(IC50).

Article Snippet: Peptide substrates for LH2 (IKGIKGIKG, abbreviated as [IKG] 3 ) and L230 (GTKGETGLKGII, abbreviated as GI-12) were ordered from LifeTein (Somerset, NJ) and the purity was determined to be greater than 98% by HPLC.

Techniques: Inhibition, Activity Assay, Control