Journal: The FASEB Journal

Article Title: Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca 2+ ] homeostasis in human autosomal‐dominant polycystic kidney disease cells

doi: 10.1096/fj.201701535rr

Figure Lengend Snippet: Figure 9. TRPV4 deglycosylation is associated with reduced channel activity. A) Summary graph of whole-cell TRPV4- dependent current-voltage (I-V) relations upon channel over- expression in CHO cells in control and after pretreatment with tunicamycin (5 mg/ml) for 24 h to block glycosylation. Currents were induced by the application of hypotonic (220 mOsm) medium from isotonic control values (300 mOsm). B) Representative Western blot of whole-cell lysates in CHO cells that overexpress TRPV4 in control and after tunicamycin treatment. Each line represents individual transfection. Lysates were probed with anti-TRPV4 and anti–b-actin Abs. g, glycosy- lated form of TRPV4.

Article Snippet: TRPV4 expression in Chinese hamster ovary cells Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Over Expression, Control, Blocking Assay, Glycoproteomics, Western Blot, Transfection

Journal: Science advances

Article Title: Control of striatal circuit development by the chromatin regulator Zswim6 .

doi: 10.1126/sciadv.adq6663

Figure Lengend Snippet: Fig. 1. ZSWIM6 localizes to the nucleus, interacts with chromatin, and regulates chromatin accessibility. (A) Experimental schematic and image of AAV-hSyn-GFP and AAV-Ef1α-Flag-Zswim6 injection into dorsal striatum. Scale bar, 100 μm. (B) Confocal images showing Flag-ZSWIM6, cell-filling GFP, and nuclear CTIP2 in striatum. Orthogonal views confirm nuclear ZSWIM6 localization. Scale bar, 10 μm. (C) Individual labeled SPN demonstrating nuclear localization of Flag-ZSWIM6 (left) and corre- sponding line plot of fluorescent intensity (right) along the line indicated in the merged image. Scale bar, 4 μm. (D) Quantification of nuclear and cytoplasmic percentage of total Flag-ZSWIM6 in labeled SPN somas (means ± SEM, n = 38 cells from three animals, ***P < 1 × 10−12, paired t test). (E) Control and Zswim6*-ΔNLS AAV construct (left). DIV9 neurons transduced with Flag-ZSWIM6 and Flag-ZSWIM6*-ΔNLS AAV and stained for MAP2, H2AZ, and Flag. Scale bar, 5 μm. (F) Flag intensity (nucleus/cyto- plasm) in control vs ΔNLS neurons (n = 29/28 cells, ***P < 1 × 10−12, paired t test). (G) Western blot following 100 to 300 mM salt extraction from HEK 293 T nuclear lysates with Flag-ZSWIM6 (n = 3). (H) Co-IP of SUZ12, HDAC1, and H3 with Flag-ZSWIM6 from HEK 293T nuclear lysates compared to untransfected control (n = 3). (I) Zswim6 SPN cKO breeding scheme (top). Images from BaseScope In Situ Hybridization (ISH) for Drd1 and Zswim6 mRNA in WT/cKO (left, scale bar: 20 μm) and quantification (right, n = 305/535 from one of two animal, ***P < 1 × 10−12, t test). (J) ATAC-seq schematic (top). Heatmaps (left) and summary graph (top, right) demonstrating normalized reads surrounding TSS in Dlx WT versus cKO P8 striatum. Box and whisker plots displaying fold change at TSS (****P < 1 × 10−12, t test). (K) Reads per million mapped reads within enhancer regions from Dlx WT versus cKO P8 striatum (top). Box and whisker plots displaying fold change within enhancer regions (****P < 1 × 10−12, t test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Frozen pellets of ~25 M HEK cells (American Type Culture Collection, CRL- 11268) (untransfected and transfected with Flag- Zswim6) were resuspended in 3 ml of lysis buffer 1 [50 mM Hepes- KOH (pH 7.5 to 8), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP- 40, 0.25% Triton X- 100, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1X cOmplete protease inhibitor, and 1X PhosSTOP phosphatase inhibitor], rotated 10 min at 4C, and pelleted by centrifuging for 5 min at 1350g, 4°C.

Techniques: Injection, Labeling, Control, Construct, Transduction, Staining, Western Blot, Extraction, Co-Immunoprecipitation Assay, In Situ Hybridization, Whisker Assay

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: HORMAD1 is phosphorylated at Ser 375 on unsynapsed chromosomes.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques:

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) Testis nuclear extracts were immunoprecipitated with the anti-SMC3 antibody, followed by treatment with (+) or without (−) phosphatase (PPase) and phosphatase inhibitors (Inhibitor). 80% of the immunoprecipitated SMC3 and the rest were separated on a gradient gel and immunoblotted with antibodies against the Ser 1083 -phosphorylated form of SMC3 (pS1083) and normal SMC3, respectively. (B) Testis nuclear extracts were immunoprecipitated without (Mock) or with the anti-pS1083 antibody. The immunoprecipitates were probed with antibodies against meiotic chromosome axis components. The asterisk marks a non-specific band. (C) Nuclear spreads of spermatocytes were labeled with anti-pS1083, anti-SYCP3 and anti-HORMAD1 antibodies. Arrowheads indicate the XY bivalent. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Immunoprecipitation, Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A and D) The insoluble fraction of testis nuclear extracts was prepared from Atm −/− (A) and Brca1 Δ11/Δ11 Trp53 +/− ( Brca1 Δ ) (D) males and probed with antibodies against meiotic chromosome axis components. (B and E) Testis nuclear extracts from Atm −/− (B) and Brca1 Δ11/Δ11 Trp53 +/− (E) males were immunoprecipitated with the anti-HORMAD1 antibody. 80% of the immunoprecipitated HORMAD1 and the rest were separated on a gradient gel and immunoblotted with anti-pS375 and anti-HORMAD1 antibodies, respectively. The asterisk marks a non-specific band. (C and F) Testis nuclear extracts from Atm −/− (C) and Brca1 Δ11/Δ11 Trp53 +/− (F) males were immunoprecipitated with the anti-SMC3 antibody. 80% of the immunoprecipitated SMC3 and the rest were separated on a gradient gel and immunoblotted with anti-pS1083 and anti-SMC3 antibodies, respectively. (G) Nuclear spreads of Brca1 Δ11/Δ11 Trp53 +/− pachytene spermatocytes were labeled with anti-pS375, anti-SYCP3 and anti-SYCP1 antibodies. Arrowheads indicate the XY bivalent. Bar, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Immunoprecipitation, Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) The insoluble fraction of testis nuclear extracts was probed with antibodies against meiotic chromosome axis components. (B) The Ser 375 -phosphorylated form of HORMAD1 was examined as in . (C) The Ser 1083 -phosphorylated form of SMC3 was examined as in . (D) Nuclear spreads of Spo11 −/− zygotene-like spermatocytes were labeled with anti-pS375, anti-SYCP3 and anti-HORMAD1 antibodies. (E) Nuclear spreads of Spo11 −/− zygotene-like spermatocytes were labeled with anti-pS1083, anti-SYCP3 and anti-HORMAD1 antibodies. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) The insoluble fraction of testis nuclear extracts was probed with antibodies against meiotic chromosome axis components. (B) The Ser 375 -phosphorylated form of HORMAD1 was examined as in . (C) The Ser 1083 -phosphorylated form of SMC3 was examined as in . (D) Nuclear spreads of Sycp3 −/− zygotene-like spermatocytes were labeled with anti-pS375, anti-SYCP1 and anti-HORMAD1 antibodies. (E) Nuclear spreads of Sycp3 −/− zygotene-like spermatocytes were labeled with anti-pS1083, anti-SYCP1 and anti-HORMAD1 antibodies. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Labeling

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A–C) Nuclear spreads of wild-type (A), Sycp3 −/− (B) and Spo11 −/− (C) zygotene-like spermatocytes were labeled with anti-γH2AX, anti-HORMAD1 and anti-SYCP1 antibodies. (D–G) Nuclear spreads of wild-type (D), Sycp3 −/− (E), Sycp1 −/− (F) and Tex12 −/− (G) zygotene-like spermatocytes were labeled with anti-γH2AX, anti-REC8 and anti-ATR antibodies. Arrowheads indicate the position of the pseudo-sex body-like staining of γH2AX. Bars, 10 µm.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Labeling, Staining

Journal: PLoS Genetics

Article Title: Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis

doi: 10.1371/journal.pgen.1002485

Figure Lengend Snippet: (A) Schematic representation of the model for regulation of phosphorylation of meiotic chromosomal proteins at S/T-Q motifs. In response to SPO11-formed DSBs (arrow 1), ATM phosphorylates histone H2AX (arrow 2) and ATR phosphorylates HORMAD1/2 (arrow 3) and SMC3 (arrow 4). Phosphorylated HORMAD1/2 serves as a marker for unsynapsis and contributes to the correct localization of ATR at unsynapsed chromosomal regions (arrow 5). At the unsynapsed chromosomes, ATR phosphorylates H2AX to promote MSUC (arrow 6), as well as HORMAD1/2 (arrow 7) and SMC3 (arrow 8). Phosphorylated HORMAD1/2 further stabilizes ATR (arrow 9) at unsynapsed chromosomes and ATR further phosphorylates HORMAD1/2 (arrow 10), amplifying the unsynapsis signal via the positive feedback loop (arrow 9 and 10). (B) The status of chromosome synapsis can be indicated by the presence or absence of HORMAD1/2 and phosphorylation of HORMAD1 and SMC3. At unsynapsed chromosomal regions, the chromosome axis contains the S/T-Q motif-phosphorylated forms of HORMAD1/2 and SMC3. When homologs are synapsed, HORMAD1/2 and the Ser 1083 -phosphorylated form of SMC3 are displaced from the chromosome axis. After desynapsis, HORMAD1/2 is again included in the chromosome axis but HORMAD1 (and possibly HORMAD2) is not phosphorylated at the S/T-Q motif. Distribution of the phosphorylated forms of other components of the chromosome axis remains to be determined.

Article Snippet: The following antibodies were also used: guinea pig anti-SYCP2, anti-SMC1β, anti-STAG3, anti-REC8 and anti-SYCP1 antibodies ; guinea pig anti-HORMAD1antibody ; rabbit anti-HORMAD1 antibody (13917-1-AP) from Proteintech Group; rabbit anti-pS/T-Q antibody (#2851) from Cell Signaling Technology; rabbit anti-pS1083 antibodies (A300-480A and IHC-00070) from Bethyl Laboratories; mouse and rabbit anti-γH2AX antibodies (#05-636 and #07-164) from Millipore; mouse anti-SYCP3 (sc-74569), rabbit anti-HORMAD2 (sc-82192), goat anti-SMC3 (sc-8198) and goat anti-ATR (sc-1887) antibodies from Santa Cruz Biotechnology; rabbit anti-SMC3 (ab9263) and rabbit anti-SYCP1 (ab15090) antibodies from Abcam; mouse anti-SYCP1 antibody (a gift from C. Heyting).

Techniques: Phospho-proteomics, Marker