Journal: Journal of Biological Chemistry

Article Title: Involvement of Protein Kinase Cδ/Extracellular Signal-regulated Kinase/Poly(ADP-ribose) Polymerase-1 (PARP-1) Signaling Pathway in Histamine-induced Up-regulation of Histamine H1 Receptor Gene Expression in HeLa Cells

doi: 10.1074/jbc.m111.253104

Figure Lengend Snippet: FIGURE 3. Effect of the PKC expression level on histamine-induced up-regulation of H1R mRNA expression. A and B, the expression plasmids encoding Ds-Red-PKC were transfected into the attached HeLa cells. After 24 h of transfection, HeLa cells were serum-starved for 24 h and then treated with 100 M histamine for 3 h. In A, total cell lysates were isolated, and the expression levels of Ds-Red-PKC and endogenous PKC were analyzed by immunoblot analysis. In B, the cells were harvested with 700 l of RNAiso Plus, and total RNA was isolated. The H1R mRNA levels were determined by real-time quantitative RT-PCR. Data are presented as the means S.E. (n 4). *, p 0.05 versus control, histamine (); **, p 0.05 versus control, Ds-Red-PKC (); #, p 0.05 versus control, histamine (). C and D, HeLa cells were reverse-transfected with 7 nM PKC-specific siRNA or 21 nM control siRNA. Twenty-four hours after transfection, the cells were starved for 24 h followed by treatment with histamine (100 M). In C, the PKC mRNA levels were determined by RT-PCR using the primers used for the cloning of PKC cDNA (top). The values are presented as the means S.E. (n 3). *, p 0.05 versus control. In D, the H1R mRNA levels were determined by real-time quantitative RT-PCR. Data are presented as the means S.E. (n 4). *, p 0.05 versus control, histamine (); #, p 0.05 versus control, histamine ().

Article Snippet: HeLa cells cultured in 12-well culture plates were cotransfected with pH1R and the internal control plasmid pRL-MPK (Promega) in a ratio of 100:1 using the PolyFect transfection reagent (Qiagen) according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Isolation, Western Blot, Quantitative RT-PCR, Control, Reverse Transcription Polymerase Chain Reaction, Cloning

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: Distribution of expressed ZBED4 in HEK293. (A) ZBED4 expression construct. (B) Immunocytochemical detection of the ZBED4 protein after transient transfection of the expression vector into HEK293 cells. Cells were double stained with antibodies against ZBED4 and Xpress and the resultant images were merged. Nuclear localization of expressed ZBED4 was detected by both antibodies and confirmed by staining with DAPI. (C) Western blot of nuclear and cytoplasmic extracts of HEK293 cells transfected for 24 or 72 hours. The blot was hybridized with anti-Xpress antibody. The expressed protein was present in the nuclear extract, confirming its immunocytochemical localization. Lanes 1 and 3: nuclear extracts; lanes 2 and 4: cytosolic extracts; lanes 1 and 2: extracts obtained after transfecting cells for 24 hours; lanes 3 and 4: extracts obtained after transfecting cells for 72 hours. (D) Localization of ZBED4 protein in Y79 cells. Note the granular pattern of ZBED4 staining in the nuclei.

Article Snippet: Y79 retinoblastoma (Y79) and human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown and transfected as previously described, 10 using the pcDNA4/HisMax-ZBED4 expression construct and a transfection reagent (PolyFect Transfection Reagent; Qiagen).

Techniques: Expressing, Construct, Transfection, Plasmid Preparation, Staining, Western Blot

Journal:

Article Title: ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

doi: 10.1167/iovs.08-2751

Figure Lengend Snippet: Specificity of anti-ZBED4 antibodies. (A) Protein extracts from both thymus and retina were separated on a Tris/Tricine-buffered 6% acrylamide/3% cross-linking gel and transferred onto PVDF membranes. Blots were incubated with the N terminus ZBED4 antibody (left) at a 1:3000 dilution. For the competition reaction (right), the antibody was incubated overnight with a 3000 molar excess of the peptide used to generate it. The labeled band that corresponds to ZBED4, apparent molecular mass of 135 kDa, is absent in the anti-ZBED+peptide blot. Similar results were obtained with the C terminus ZBED4 antibody. (B, C) Y79 cells were transiently transfected with siRNA duplexes targeted to the 5′ or 3′ regions of ZBED4 mRNA. (B) Ninety-six hours after transfection, total RNA was extracted, reverse transcribed, and subjected to QPCR. The level of mRNA expression in all samples is relative to that in control cells (C) and shows the silencing of ZBED4 in HEK 293 cells after transiently transfecting all three siRNAs. ZBED4 antibodies barely detected or did not detect at all the expression of ZBED4 in the silenced samples. C, control cells; TC, cells with transfection reagent only; NC, negative control (totally unrelated to ZBED4 siRNA); S1, S2, S3, double-stranded ZBED4 RNA oligomers (siRNAs). (C) Protein extracts from transfected HEK 293 cell lysates were assayed for ZBED4 gene silencing by Western blot analysis.

Article Snippet: Y79 retinoblastoma (Y79) and human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown and transfected as previously described, 10 using the pcDNA4/HisMax-ZBED4 expression construct and a transfection reagent (PolyFect Transfection Reagent; Qiagen).

Techniques: Incubation, Labeling, Transfection, Reverse Transcription, Expressing, Control, Negative Control, Western Blot