osbpl5 antibody  (Bio-Techne corporation)

Journal: Life Science Alliance

Article Title: Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

doi: 10.26508/lsa.201900431

Figure Lengend Snippet: (A) OSBPL5 and OSBPL8 are paralogs that encode ORP5 and ORP8. These proteins are lipid transporters involved in lipid counter transport between the ER and the PM; they specifically exchange PtdSer in the ER with PI4P in the PM. The driving force of this process is a PI4P concentration gradient, whereby PI4P levels are high in the PM and are kept low at the ER by the action of the SAC1 phosphatase which immediately hydrolyzes PI4P. (B) RNAi knockdown screen of OSBPL5 , OSBPL8 , and SAC1P orthologs in an activated let-60 C. elegans . RNAi was induced by feeding let-60(n1046) L1 worms through adult stage with E. coli strain HT115, producing dsRNA to target genes. The presence of the MUV phenotype was scored using Differential Interface Contrast (DIC)/Nomarski microscopy. Previous reports show that h eo-1 and riok-1 potently suppress the let-60 G13D MUV phenotype and, hence, were used as positive controls (**** P < 0.0001, * P < 0.05). OSBPL , oxysterol-binding protein like; obr , oxysterol-binding protein related; SAC1P, SAC1-like phosphatidylinositide phosphatase.

Article Snippet: Anti-osbpl5 (NB100-57071) and YAP-1 (NB110-58358) antibodies were purchased from Novus.

Techniques: Concentration Assay, Knockdown, Microscopy, Binding Assay

Journal: Life Science Alliance

Article Title: Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

doi: 10.26508/lsa.201900431

Figure Lengend Snippet: (A) shRNA knockdown of ORP5 and ORP8 separately and simultaneously in MCF-7 breast cancer cells was validated by Western blotting, and β-actin levels were used as loading controls. (B) Parental (WT) and knockdown cells were transiently transfected with GFP-KRASG12V and mCherryCAAX (an endomembrane marker) or GFP-LactC2 and mCherryCAAX and imaged in a confocal microscope. Representative images are shown. (C) The extent of KRAS and LactC2 mislocalization was quantified using Manders coefficient, which measures the extent of colocalization/overlap of GFP and mCherry signals. Significant differences were evaluated using t tests (±SEM, n ≥ 6) (* P < 0.05, ** P < 0.01, *** P < 0.001); scale bar 20 μm.

Article Snippet: Anti-osbpl5 (NB100-57071) and YAP-1 (NB110-58358) antibodies were purchased from Novus.

Techniques: shRNA, Knockdown, Western Blot, Transfection, Marker, Microscopy

Journal: Life Science Alliance

Article Title: Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

doi: 10.26508/lsa.201900431

Figure Lengend Snippet: shRNA knockdown of ORP5 and ORP8 separately and simultaneously in BxPC-3, PANC-1, MOH, and MiaPaCa-2 cells was validated by Western blotting, and β-actin levels were used as loading controls.

Article Snippet: Anti-osbpl5 (NB100-57071) and YAP-1 (NB110-58358) antibodies were purchased from Novus.

Techniques: shRNA, Knockdown, Western Blot

Journal: Life Science Alliance

Article Title: Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

doi: 10.26508/lsa.201900431

Figure Lengend Snippet: ORP5 and ORP8 were knocked down separately and simultaneously by shRNA in BxPC-3, PANC-1, MiaPaCa-2, and MOH cells. Parental and knockdown cells were grown in six-well plates for 5 d and counted every day. Cell numbers of each cell line at day 5 were normalized to their cell number at 24 h and plotted. Significant differences were evaluated using t tests (±SEM, n = 3) (** P < 0.01, *** P < 0.001).

Article Snippet: Anti-osbpl5 (NB100-57071) and YAP-1 (NB110-58358) antibodies were purchased from Novus.

Techniques: shRNA, Knockdown

Journal: Life Science Alliance

Article Title: Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

doi: 10.26508/lsa.201900431

Figure Lengend Snippet: ORP5 and ORP8 were knocked down separately and simultaneously by shRNA in BxPC-3, PANC-1, MiaPaCa-2, and MOH cells. Parental and knockdown cells were seeded in soft agar, with a base layer of 1% agar–media mixture and a top layer of a 0.6% agar–cell suspension mix in six-well plates. After 2–3 wk, the colonies were stained with 0.01% crystal violet and imaged. Colony numbers were quantified by ImageJ and significant differences were evaluated using t tests (±SEM, n = 3) (* P < 0.05, ** P < 0.01).

Article Snippet: Anti-osbpl5 (NB100-57071) and YAP-1 (NB110-58358) antibodies were purchased from Novus.

Techniques: shRNA, Knockdown, Suspension, Staining

Journal: Life Science Alliance

Article Title: Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

doi: 10.26508/lsa.201900431

Figure Lengend Snippet: (A) shRNA knockdown of ORP5 and ORP8 separately and simultaneously in HCT116 isogenic cell lines was validated by Western blotting, and β-actin levels were used as loading controls. (B) Parental and ORP5 knockdown cells of HCT116 −/WT and HCT116 Mut/WT cells were blotted for ORP8 to evaluate changes in protein expression level upon ORP5 knockdown, and β-actin levels were used as loading controls. −/WT: cells with a single KRAS WT allele, Mut/WT: cells with one KRAS-mutant allele and one KRAS WT allele.

Article Snippet: Anti-osbpl5 (NB100-57071) and YAP-1 (NB110-58358) antibodies were purchased from Novus.

Techniques: shRNA, Knockdown, Western Blot, Expressing, Mutagenesis

Journal: Life Science Alliance

Article Title: Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

doi: 10.26508/lsa.201900431

Figure Lengend Snippet: (A, B, C) Box plots indicating quartiles of ORP5 mRNA expression level in patient samples in cohorts of pancreatic cancer (GDC TCGA PAAD, n = 223), lung cancer (TCGA LUNG, n = 1,299), and of 33 types of cancer (GDC Pan-Cancer [PANCAN], n = 20,163) with or without KRAS mutations. Statistical significance was analyzed with Welch’s t test. Kaplan–Meier survival plots based on expression levels of ORP5 (B) and ORP8 (C) in cohorts listed in (A). Plots were generated using the University of California, Santa Cruz (UCSC) Xena Browser.

Article Snippet: Anti-osbpl5 (NB100-57071) and YAP-1 (NB110-58358) antibodies were purchased from Novus.

Techniques: Expressing, Generated

Journal: Cancer science

Article Title: Oxysterol binding protein-related protein-5 is related to invasion and poor prognosis in pancreatic cancer.

doi: 10.1111/j.1349-7006.2008.00987.x

Figure Lengend Snippet: Fig. 2. Expression levels of oxysterol binding protein-related protein (ORP)-5 in hamster and human pancreatic cancer cell lines. PC1 was hamster pancreatic cancer cell line with a low potential for invasion and metastasis, and PC1.0 was hamster pancreatic cancer cell line with a high potential for invasion and metastasis. (a) ORP5 expression in the hamster pancreatic cancer cell lines PC1.0 and PC1 after transfection of short interfering RNA (siRNA) or expression vector. ORP5 was expressed at a high level in the PC1.0 cells and at a low level in the PC1 cells, at both the mRNA and protein levels. Transfection of ORP5 siRNA into PC1.0 cells resulted in a significant decrease in the expression level of ORP5 at 48–72 h after transfection. Transfection of pcDNA/hamORP5 into PC1 cells resulted in a significant increase in the expression level of ORP5 at 24–72 h after transfection. (b) The expression levels of ORP5 in human pancreatic cancer cell lines. At both the mRNA and protein level, ORP5 was expressed at a high level in Capan1, Capan2, and Panc1 cells, at a moderate level in the MiaPaCa2 cells, and at a low level in the Hs700T cells. (c) ORP5 expression in the human pancreatic cancer cell lines Capan2 and Hs700T after transfection of siRNA or expression vector. ORP5 was expressed at a high level in the Capan2 cells, but at a low level in the Hs700T cells, at both the mRNA and protein levels. Transfection of ORP5 siRNA into Capan2 cells resulted in a significant decrease in the expression level of ORP5 at 48–72 h after transfection. Transfection of pcDNA/huORP5 into Hs700T cells resulted in a significant increase in the expression level of ORP5 at 24–72 h after transfection. (d) The ORP5 stable transfectant cells (PC1 + ORP5 or Hs700T + ORP5) showed high expression levels of ORP5, and the LacZ stable transfectant cells (PC1 + LacZ or Hs700T + LacZ) showed low expression levels of ORP5. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription–polymerase chain reaction.

Article Snippet: The membrane was blocked with 5% skim milk (BD, Franklin Lakes, NJ, USA) in Tris-buffered saline (TBS)–Tween 20 (0.1%) at room temperature for 1 h and then incubated with polyclonal goat anti-ORP5 antibody (Imgenex, San Diego, CA, USA), β-actin antibody (Cell Signaling Technology, Beverly, MA, USA), or V5 antibody (Invitrogen) for 1 h at room temperature.

Techniques: Expressing, Binding Assay, Transfection, Small Interfering RNA, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer science

Article Title: Oxysterol binding protein-related protein-5 is related to invasion and poor prognosis in pancreatic cancer.

doi: 10.1111/j.1349-7006.2008.00987.x

Figure Lengend Snippet: Fig. 4. Immunohistochemical staining of oxysterol binding protein-related protein (ORP)-5. The exp- ression level of ORP5 in pancreatic cancer specimens was analyzed in comparison with that in the acinar cells of the pancreas. (a) ORP5-negative pancreatic cancer tissue. (b) ORP5-positive pancreatic cancer tissue. ORP5 was highly expressed in the cytoplasm of cancer cells. (c) Weak expression of ORP5 in the acinar cells of the pancreas. Scale bar = 100 μm.

Article Snippet: The membrane was blocked with 5% skim milk (BD, Franklin Lakes, NJ, USA) in Tris-buffered saline (TBS)–Tween 20 (0.1%) at room temperature for 1 h and then incubated with polyclonal goat anti-ORP5 antibody (Imgenex, San Diego, CA, USA), β-actin antibody (Cell Signaling Technology, Beverly, MA, USA), or V5 antibody (Invitrogen) for 1 h at room temperature.

Techniques: Immunohistochemical staining, Staining, Binding Assay, Comparison, Expressing