Journal: Molecular Cancer

Article Title: MutS homologue hMSH5: role in cisplatin-induced DNA damage response

doi: 10.1186/1476-4598-11-10

Figure Lengend Snippet: Analysis of cisplatin-induced hMSH5 association with chromatin and nuclear foci formation . ( A ) 293T/f-hMSH5 and 293T/f-hMSH5 Y742F cells were treated with 20 μM cisplatin, and cross-linked bulk chromatin was immunoprecipitated by an α-acetyl-histone H3 antibody 5 hrs post-treatment. The levels of chromatin-associated hMSH5 and its levels of phosphorylation were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Equal levels of acetyl-histone H3 and histone H4 were present in the immunoprecipitates. ( B ) Analysis of hMSH4 chromatin association following cisplatin treatment. Chromatin was prepared from 293T/f45 cells treated with cisplatin. The levels of chromatin-associated hMSH5 and hMSH4 were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Mouse IgG was used as a negative control. kDa , molecular weight ( Mr ) in thousands. ( C ) Examination of γ-H2AX foci formation 24 hrs post cisplatin exposure. 293T, 293T/f-hMSH5, 293T/f-hMSH5 Y742F , and 293T cells subjected to hMSH5 RNAi were used for this analysis. Cells possessing greater than 15 foci/nucleus were graphically displayed. ( D ) Immunoblotting analysis of the effectiveness of hRad51 or hMSH5 knockdown in 293T cells transfected with pmH1P-Bsd/hRad51 sh-1 or pmH1P-Bsd/hMSH5 sh-2. α-Tubulin was used as a loading control. kDa , molecular weight ( Mr ) in thousands. ( E ) Analysis of hMSH5 and hRad51 knockdown on cisplatin-induced hRad51 and hMSH5 nuclear foci formation. Cells were subjected to 10 μM cisplatin for 2 hrs and were analyzed for hMSH5 foci formation 6 hrs post cisplatin removal. Cells that possessed five or more nuclear foci for hMSH5 or hRad51 were scored. Error bars represent standard deviations of the means of three independent measurements. Statistically significant differences between knockdown and control cells were indicated with asterisks (p < 0.05, Student t -test).

Article Snippet: Antibodies used in the experiments included α-acetyl histone H3 (Upstate), mouse IgG (Upstate), α-p-Tyr (Cell Signaling), α-hMSH5 [ ], α-hMSH4 [ ], and α-histone H4 (Cell Signaling).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Molecular Weight, Transfection

Journal: Endocrinology

Article Title: Essential Roles of Epithelial Bone Morphogenetic Protein Signaling During Prostatic Development

doi: 10.1210/en.2013-2054

Figure Lengend Snippet: Decreased expression of Nkx3.1 in the Bmpr1a -CKO mutants. A and B, Nkx3.1 expression decreased in mutant prostate at P1 (A) and in the mutant AP at P28 (B). The relative mRNA equivalents for each sample were normalized by the RNA levels for ribosomal protein L8. Bars represent the mean ± SE of triplicate assays of RNA from pooled tissues (A) and 6 tissue samples (B). Statistical significance was indicated by asterisks (*, P < .05). C, Nkx3.1 was detected in the luminal epithelia of the control AP at P28. D, Significantly reduced levels of Nkx3.1 protein were detected in the mutant AP. E, The ratios of Nkx3.1-positive cells were shown in the graph. F, Colocalization of Nkx3.1 and Bmpr1a in the AP luminal epithelia of adult mice. G, Colocalization of pSmad1/5/8 and Nkx3.1 in the AP luminal epithelia of adult mice. F and G, Cryosections were used. Scale bars, 20 μm. H, Genomic sequences of the mouse Nkx3.1 aligned with its orthologous loci in human and opossum. The sequence alignment was performed using MultiPipMaker. A noncoding region conserved from human to opossum was indicated with black boxes in the C1 and C2 regions. The black arrow indicated exons of mouse Nkx3.1 . Coding and untranslated sequences were shaded with red and yellow, respectively. A 5-kb (5399 base) region in the 3′-genomic region of Nkx3.1 contained a candidate enhancer region for the mouse prostate. The scale at the bottom of the alignment indicated relative positions in the mouse Nkx3.1 locus. I, The candidate 5-kb prostatic regulatory enhancer activated expression of a luciferase reporter in response to Smad1/4 expression (by 6 independent assays). It also responded to the addition of Bmp7 (by 3 independent assays) (means ± SE) (*, P < .05). a, Control. b, Transfected with Smad1/4 gene. c, Control. d, Transfected with Smad1/4 gene + addition of Bmp7. J, ChIP/PCR assay on bladder neck of ICR mice including prostate region at P2 showed pSmad1/5/8 binding to regions of C1 and C2 in the 3′-region of mouse Nkx3.1 . Both regions were enriched in chromatin immunoprecipitated with antiacetylated histone H3 as a positive control.

Article Snippet: The bladder-neck region containing the prostate of ICR mice was dissected from the pups at postnatal day 2. pSmad1/5/8 (Cell Signaling Technology) and acetyl-histone H3 (Upstate) antibodies (2 μg) were used for immunoprecipitation.

Techniques: Expressing, Mutagenesis, Genomic Sequencing, Sequencing, Luciferase, Transfection, Binding Assay, Immunoprecipitation, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency

doi: 10.1074/jbc.M112.342220

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: All other reagents were purchased from Sigma, Bio-Rad, and Invitrogen. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Provider Catalog no. Dilutions used in immunoblotting M-opsin Dr. Cheryl Craft (Keck School of Medicine) 1:2000 Cone arrestin (CAR) Dr. Cheryl Craft (Keck School of Medicine) 1:2000 S-opsin Dr. Muna Naash (University of Oklahoma Health Sciences Center) 1:1000 CNGA3 Custom antibody generated by YenZym Antibodies, LLC (Ref. 25 ) 1:250 Gnat2 Santa Cruz Biotechnology sc-390 1:500 GADD 153 (CHOP-10) Santa Cruz Biotechnology sc-575 1:100 Phospho-eIF2α Cell Signaling Technology 3398 1:500 Phospho-IP 3 R Cell Signaling Technology 3760 1:250 Caspase-7 Cell Signaling Technology 9492 1:250 Caspase-12 Cell Signaling Technology 2202 1:250 Endo G Cell Signaling Technology 4969 1:250 AIF Cell Signaling Technology 4642 1:500 Cytochrome c Cell Signaling Technology 4272 1:250 Caspase-9 Cell Signaling Technology 9504 1:250 Caspase-3 Cell Signaling Technology 9661 1:250 Calpain II Cell Signaling Technology 2539 1:500 Bcl-2 Epitomic 1017-1 1:250 Bcl-2-x L Epitomic 1018-1 1:250 Calpain I Abcam ab28258 1:500 Grp78/BiP Abcam ab21685 1:500 β-Actin Abcam ab-6276 1:2000 Acetyl-histone H3 (H3) Upstate Cell Signaling Solution 07-540 1:2000 TATA binding protein Thermo Scientific, Inc. MA1-10883 1:500 Open in a separate window List of antibodies used in this study Recordings of Electroretinograms (ERG) Full-field ERG recordings were carried out as described previously ( 16 ).

Techniques: Western Blot, CRAfT Assay, Generated, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency

doi: 10.1074/jbc.M112.342220

Figure Lengend Snippet: Enhanced expression of the ER stress marker proteins in CNGA3−/−/Nrl−/− and CNGB3−/−/Nrl−/− retinas. The expression levels of Grp78/Bip, phospho-eIF2α, and CHOP were examined in CNGA3−/−/Nrl−/−, CNGB3−/−/Nrl−/−, and Nrl−/− mice at P30. A, left panel: shown are representative images of the Western blot detections. Total retinal protein lysate was used for detection of Grp78/Bip and phospho-eIF2α (actin was used as a loading control, upper three panels), and retinal nuclear preparation was used for detection of CHOP. (H3 was used as a loading control, lower two panels.) Right panel: densitometric analysis of the relative expression levels of phospho-eIF2α in CNGA3−/−/Nrl−/−, CNGB3−/−/Nrl−/−, and Nrl−/− retinas. Data are represented as means ± S.E. of measurements from three to four independent experiments using retinas from four to five mice. Unpaired Student's t test was used for determination of the significance (*, p < 0.05). B, CHOP activation in CNG channel-deficient retina. Shown are images of CHOP staining on retinal sections of Nrl−/− (a) and CNGA3−/−/Nrl−/− (b) mice. C, co-localization of Grp78/Bip with S-opsin in CNG channel-deficient retina. Co-labeling was performed using rabbit anti-Grp78/Bip and goat anti-S-opsin antibodies. Shown are images of the co-labeling on retinal sections of WT (a–c), CNGA3−/− (d–f), and CNGB3−/− (g–i) mice with higher magnification images alongside (c′, f′, i′). IS, inner segment; ONL, outer nuclear layer; IB, immunoblot. Scale bar, 20 μm.

Article Snippet: All other reagents were purchased from Sigma, Bio-Rad, and Invitrogen. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Provider Catalog no. Dilutions used in immunoblotting M-opsin Dr. Cheryl Craft (Keck School of Medicine) 1:2000 Cone arrestin (CAR) Dr. Cheryl Craft (Keck School of Medicine) 1:2000 S-opsin Dr. Muna Naash (University of Oklahoma Health Sciences Center) 1:1000 CNGA3 Custom antibody generated by YenZym Antibodies, LLC (Ref. 25 ) 1:250 Gnat2 Santa Cruz Biotechnology sc-390 1:500 GADD 153 (CHOP-10) Santa Cruz Biotechnology sc-575 1:100 Phospho-eIF2α Cell Signaling Technology 3398 1:500 Phospho-IP 3 R Cell Signaling Technology 3760 1:250 Caspase-7 Cell Signaling Technology 9492 1:250 Caspase-12 Cell Signaling Technology 2202 1:250 Endo G Cell Signaling Technology 4969 1:250 AIF Cell Signaling Technology 4642 1:500 Cytochrome c Cell Signaling Technology 4272 1:250 Caspase-9 Cell Signaling Technology 9504 1:250 Caspase-3 Cell Signaling Technology 9661 1:250 Calpain II Cell Signaling Technology 2539 1:500 Bcl-2 Epitomic 1017-1 1:250 Bcl-2-x L Epitomic 1018-1 1:250 Calpain I Abcam ab28258 1:500 Grp78/BiP Abcam ab21685 1:500 β-Actin Abcam ab-6276 1:2000 Acetyl-histone H3 (H3) Upstate Cell Signaling Solution 07-540 1:2000 TATA binding protein Thermo Scientific, Inc. MA1-10883 1:500 Open in a separate window List of antibodies used in this study Recordings of Electroretinograms (ERG) Full-field ERG recordings were carried out as described previously ( 16 ).

Techniques: Expressing, Marker, Western Blot, Activation Assay, Staining, Labeling