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

Journal: Experimental and Therapeutic Medicine

Article Title: LSKL peptide alleviates subarachnoid fibrosis and hydrocephalus by inhibiting TSP1-mediated TGF-β1 signaling activity following subarachnoid hemorrhage in rats

doi: 10.3892/etm.2016.3640

Figure Lengend Snippet: LSKL peptide inhibited TSP1-mediated TGF-β1 signaling activity following SAH. Quantitative analyses of (A) TSP1 and (B) total TGF-β1 and active TGF-β1 in the CSF on days 3–5 after SAH. (C) Ratio of active TGF-β1 to total TGF-β1 in the CSF on days 3–5 after SAH. (D) Representative western blot bands of p-Smad2/3 and (E) quantitative analyses of p-Smad2/3 expression on day 5 after SAH. Relative densities of each protein have been normalized against the sham group. Data are expressed as the mean ± standard error of the mean (n=10). *P<0.05 vs. the sham group; # P<0.05 vs. the SAH+PBS group. SAH, subarachnoid hemorrhage; CSF, cerebrospinal fluid; TSP1, thrombospondin-1; LSKL, leucine-serine-lysine-leucine; PBS, phosphate buffer solution.

Article Snippet: These CSF samples were mixed and divided into three equal shares for the detection of TSP1, activated TGF-β1 and total TGF-β1 using the respective ELISA kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China).

Techniques: Activity Assay, Western Blot, Expressing

Journal: International Journal of Nanomedicine

Article Title: Docetaxel-loaded solid lipid nanoparticles suppress breast cancer cells growth with reduced myelosuppression toxicity

doi: 10.2147/IJN.S70919

Figure Lengend Snippet: Genes regulated by Taxotere ® and docetaxel-loaded solid lipid nanoparticles were confirmed by quantitative polymerase chain reaction and immunoblotting. Notes: Cell cycle-related genes of E2F8 ( A ) and OIP5 ( B ), proliferation-related genes of NASP ( C ) and SOD2 ( D ), and apoptosis-related genes of PDCD4 ( E ) and PIK3R2 ( F ) were chosen for detection by quantitative polymerase chain reaction and immunoblotting. In quantitative polymerase chain reaction detection, mock-treated cells were set as the control and samples were normalized with the control. In immunoblotting detection, β-actin was used as the loading control. Abbreviations: BSN, blank solid lipid nanoparticle; DSN, docetaxel-loaded solid lipid nanoparticle; GLU, glucose; qPCR, quantitative polymerase chain reaction; TAX, Taxotere.

Article Snippet: Primary antibodies of rabbit anti-β-actin, E2f8, MRE11A, ERBB3, IGFBP6, ATF3, CCNG2, SOD2, IGFBP3, CADM1, PDCD4, GADD45A, and MKI67 were purchased from Beijing Biosynthesis Technology Co., Ltd., (Beijing, People’s Republic of China), rabbit anti-MCM6, OIP5, and NASP were purchased from Proteintech Group, Inc., (Chicago, IL, USA), rabbit anti-FAM172A and MYB were purchased from Abgent, Inc. (San Diego, CA, USA) and rabbit anti-ATRX was purchased from GeneTex, Inc. (Irvine, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Nanomedicine

Article Title: Docetaxel-loaded solid lipid nanoparticles suppress breast cancer cells growth with reduced myelosuppression toxicity

doi: 10.2147/IJN.S70919

Figure Lengend Snippet: Primers used for quantitative polymerase chain reaction

Article Snippet: Primary antibodies of rabbit anti-β-actin, E2f8, MRE11A, ERBB3, IGFBP6, ATF3, CCNG2, SOD2, IGFBP3, CADM1, PDCD4, GADD45A, and MKI67 were purchased from Beijing Biosynthesis Technology Co., Ltd., (Beijing, People’s Republic of China), rabbit anti-MCM6, OIP5, and NASP were purchased from Proteintech Group, Inc., (Chicago, IL, USA), rabbit anti-FAM172A and MYB were purchased from Abgent, Inc. (San Diego, CA, USA) and rabbit anti-ATRX was purchased from GeneTex, Inc. (Irvine, CA, USA).

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: Specific deletion of protein phosphatase 6 catalytic subunit in Sertoli cells leads to disruption of spermatogenesis

doi: 10.1038/s41419-021-04172-y

Figure Lengend Snippet: A β-Catenin (a marker of adherens junctions, green) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. B ZO2 (a marker of tight junctions, green) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. C TEX14 (a marker of a marker of testicular intercellular junctions, green) and β-actin (red) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. D Vimentin (a marker for Sertoli apical extensions, green) immunofluorescence of Ppp6c WT and Ppp6c cKO mice testes. Scale bar: (left) 50 μm, (right) 20 μm. Nuclei are stained with DAPI.

Article Snippet: PPP6C antibody (rabbit, A300-844A; Bethyl Laboratories, Inc.); SYCP3 antibody (rabbit, NB300-231; Novus Biologicals); α-tubulin antibody (rabbit, 2144; Cell Signaling Technology, Inc.); β-actin antibody (mouse, 3700; Cell Signaling Technology, Inc.); Phospho-β-Catenin (Ser552) (D8E11) antibody (rabbit, 5651; Cell Signaling Technology, Inc.); SYCP3 antibody (mouse, sc-74569; Santa Cruz); γH2AX antibody (rabbit, 9718; Cell Signaling Technology, Inc.); MVH antibody (mouse, ab27591; abcam); SYCP1 antibody (rabbit, ab15090; abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); β-catenin antibody (rabbit, 51067-1-AP, Proteintech); ZO2 antibody (rabbit, 18900-1-AP, Proteintech); TEX14 antibody (rabbit, 18351-1-AP, Proteintech); Vimentin antibody (rabbit, 10366-1-AP, Proteintech); HA-Tag mab (mouse, AE008; ABclonal); c-Myc antibody (mouse, m4439; sigma); green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (L21409, Thermo).

Techniques: Marker, Immunofluorescence, Staining