Journal: International Journal of Molecular Sciences

Article Title: Spinal Canal and Spinal Cord in Rat Continue to Grow Even after Sexual Maturation: Anatomical Study and Molecular Proposition

doi: 10.3390/ijms232416076

Figure Lengend Snippet: Growth factor expression in the cervical spinal cord. ( A ) Detection of FGF2, VEGFA, BDNF, and IGF1 expressions in the cervical spinal cords of a female rat at 4, 8, and 24 weeks of age using Western blot analysis. Expression of FGF2, VEGFA, and BDNF visually declined as rats grew, whereas a decrease in IGF1 expression was not apparent. ( B ) Quantification of band intensities. Results represent the mean for the relative band intensity values to GAPDH ± SEM (n = 3). The cervical spinal cord of a 4-week-old subject demonstrated significantly higher band intensity of BDNF than that of a 24-week-old subject. * p < 0.05. One-way ANOVA with the Tukey– Kramer test.

Article Snippet: Following that, membranes were blocked with 5% powdered milk (<1% fat) in PBS for 1 h at room temperature, followed by overnight incubation with primary antibodies: anti-BDNF (1:2000, rabbit from GeneTex), anti-FGF2 (1:2000, rabbit from Elabscience Biotechnology), anti-VEGFA (1:100, mouse from Novus Biologicals), anti-IGF1 (1:2000, rabbit from Bioss Antibodies), and anti-GAPDH (1:5000, rabbit from GeneTex).

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Structure prediction of TMEM95 protein (left) using IZUMO1 (right) as template, created by SWISS-MODEL software. ( B ) Tmem95 KO allele generated following CRISPR-mediated edition. CRISPR target sequence and PAM are depicted in blue and purple letters, respectively. ( C ) The deletion of 10 bp altered Tmem95 ORF. Large letters indicate the aminoacid sequence corresponding to the codons (DNA sequence) shown in smaller letters below. ( D ) Western Blot images for TMEM95, IZUMO1 and β-tubulin proteins from protein extracts from WT or KO sperm. Graph on right indicates the abundance of IZUMO1 in WT and KO extracts. ( E ) Immunocytochemistry images of KO and WT sperm stained with an antibody against TMEM95 and the acrosomal stain PNA. TMEM95 localized to the acrosomal cap in acrosome intact sperm and in the equatorial segment after acrosome reaction. ( F ) Immunocytochemistry images of acrosome intact (upper images) or reacted (lower images) WT sperm stained against IZUMO1 and TMEM95. Both proteins relocalize to the equatorial segment following acrosome reaction.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Software, Generated, CRISPR, Sequencing, Western Blot, Immunocytochemistry, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Immunoblotting of protein extracts obtained from WT and KO epididymal sperm samples following two lysis protocols. In lysis protocol #1, sperm were re-suspended in 4X reducing SDS Sample Buffer and boiled for 10 min. In lysis protocol #2, sperm were re-suspended in 1% Octyl β-D-glucopyranoside solution in PBS and incubated on ice for 30 min . Supernatants were probed with anti-TMEM95 (MyBiosource MBS7004333), anti-IZUMO1 (Abcam ab211623) or anti-β-TUBULIN (Sigma T8328) antibodies. ( B ) Gel electrophoresis of PCR products amplified from cDNA obtained from testis, seminal vesicle (S.V.) prostate (prost.), epididymis (epid.) or a negative control testis RNA not retrotranscribed (RT-) to detect Gapdh and Tmem95 transcripts. ( C ) Immunoblotting of protein extracts obtained from WT epididymal sperm, testis or accessory glands (seminal vesicle and prostate). Same antibodies than ( A ). ( D ) Uncropped images of the WB used to generate . ( E ) WB images used for the quantification of IZUMO1 and β-TUBULIN shown in . Four samples were used for quantification (marked with asterisk), as β-TUBULIN band on samples #4 was dispersed, leading to inaccurate quantification.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Western Blot, Lysis, Incubation, Nucleic Acid Electrophoresis, Amplification, Negative Control

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Immunocytochemistry images of WT sperm stained with PNA and IZUMO1 antibody (upper row) or PNA and TMEM95 antibody (lower row) in the absence of permeabilizing agents. Despite the absence of permeabilizing agents (30 min fixation in 4% PFA without Triton X-100), TMEM95 and inner acrosomal (PNA) and acrosomal membrane (IZUMO1) markers were detected. ( B ) IZUMO1 relocates to the equatorial segment following acrosome reaction in TMEM95 KO sperm. Upper row shows one acrosome intact spermatozoon. Lower rows show WT and KO acrosome reacted sperm where IZUMO1 has relocated to the equatorial segment.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Immunocytochemistry, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Binding analysis using the AVEXIS assays shows that the soluble recombinant TMEM95 ectodomain does not interact with JUNO nor with IZUMO1. The entire ectodomains of the named proteins were expressed in HEK293-6E cells either as biotinylated baits or as pentameric beta-lactamase-tagged preys. Bait proteins were immobilised on streptavidin-coated plates and captured prey proteins quantified by measuring the absorbance of a colorimetric reaction product of the beta-lactamase substrate, nitrocefin. The CD200R (bait)-CD200 (prey) binding pair was used as positive control. The same prey, CD200R, was tested against TMEM95 and is shown as negative control. Bars represent means + s.d.; n = 3. ( B ) HEK293 cells stably expressing the N-terminal half of GFP (GFP1-7) and mouse JUNO stained with a highly avid IZUMO1 probe. ( C ) HEK293 cells stably expressing the C-terminal half of GFP (GFP8-11) and mouse IZUMO1 stained with a highly avid JUNO. ( D ) TMEM95 does not induce fusion when expressed in HEK293T cells in the presence of JUNO and IZUMO1 using a GFP-complementation cell fusion assay. HEK293T cells expressing either half of GFP and either JUNO or IZUMO1 were mixed and their fusogenic ability visualized by GFP fluorescence. The IZUMOI-expressing cells were either mock transfected prior to mixing (Control), transfected with Syncitin a , as a positive fusion control, or Tmem95 . By contrast to the cells transfected with Syncytin a , Tmem95 did not induce cell fusion. Cell nuclei are stained with DAPI and scale bar represents 20 µm.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Binding Assay, Recombinant, Positive Control, Negative Control, Stable Transfection, Expressing, Staining, Cell Fusion Assay, Fluorescence, Transfection

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Amino acid sequence of FKBP1A underlined in blue fused via a short linker to the N-terminal region of GFP (GFP 1-7 ) (green underline). ( B ) Amino acid sequence of the C-terminal region of GFP (GFP 8-11 ) highlighted in green, fused to RB, underlined in blue. (C, C´, D and D´) The cells expressing the GFP1-7 stably express mouse Juno and the cells expressing the GFP8-11 stably express mouse Izumo1. To establish functional cell surface expression of both Juno and Izumo1 on the cells, noth cell lines were stained with the corresponding pentameric FLAG-tagged Izumo1 and Juno preys. The cells expressing mouse Juno were stained with the Izumo1 prey but not Juno, and the cells expressing mouse Izumo1 stained with the Juno prey but not Izumo1. Images are representative of at least three independent replicates. Nuclei are stained with DAPI (blue) and scale bars represents 10 µm.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Sequencing, Expressing, Stable Transfection, Functional Assay, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Generated, CRISPR, Recombinant, Plasmid Preparation, Sequencing

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on the interaction between the regulatory subunit and phosphorylated IRS proteins in response to IGF-1. (a) Protein and phosphorylation levels of the IGF-1 receptor in cells of each genotype. The cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 5 min. Cell lysates were subjected to immunoprecipitation with αIGF-1R followed by immunoblotting with αIGF-1R (top panel) or 4G10 (bottom panel). (b) Interaction between IRS proteins and the regulatory subunit. Cell lysates were subjected to immunoprecipitation with anti-IRS-1 (αIRS-1; left panels), anti-IRS-2 (αIRS-2; middle panels), or anti-Gab-1 (αGab-1; right panels) antibody followed by immunoblotting with the same antibody (top panels), 4G10 antibody (middle panels), or αp85pan antibody (bottom panels). Wild, wild type; hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on the PI 3-kinase activity associated with each signaling molecule. (a) PI 3-kinase activities associated with the regulatory subunits. The cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 5 min. Cell lysates were subjected to immunoprecipitation with αp85pan (left panels), αp85α (middle panels), or αp85β (right panels) antibody followed by the PI 3-kinase assay. The top panels show representative results, and in the bottom panels each bar represents the mean ± standard deviation of the relative PI-3 kinase activity calculated from the results of three independent experiments. In the αp85pan precipitation: *, P value of <0.05 for wild-type (Wild) versus null cells. In the p85β precipitation: *, P value of <0.01 for wild versus heterozygous KO (Hetero) cells; **, P value of <0.01 for wild versus null cells. (b) PI 3-kinase activities associated with the catalytic subunit and tyrosine-phosphorylated proteins. Cell lysates were subjected to immunoprecipitation with αp110α (left panels) or 4G10 (right panels) antibody followed by the PI 3-kinase assay. Top panels show representative results, and in the bottom panels each bar represents the mean ± standard deviation of the relative PI-3 kinase activity calculated from the results of three independent experiments. *, P value of <0.01 for wild versus null cells.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Immunoprecipitation, Kinase Assay, Standard Deviation

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on production of PIP3 in response to IGF-1 in vivo. (a) IGF-1-induced PIP3 production in cells of each genotype. Cells were labeled with [32P]orthophosphate as described in Materials and Methods and stimulated with 10 nM IGF-1 for the indicated period. 32P-labeled phospholipids were extracted and separated by TLC. In the graph, the mean levels of PIP3 normalized to the total labeled phospholipids from two independent experiments are shown. (b) Time course of PI 3-kinase associated with phosphotyrosine complex in cells of each genotype. Cells were stimulated with 10 nM IGF-1 for the indicated period and subjected to immunoprecipitation with 4G10 followed by the PI 3-kinase assay. (c) Expression levels of PTEN and SHIP in cells of each genotype. Cell lysates were subjected to immunoblotting with anti-PTEN (αPTEN; top panel) or anti-SHIP (αSHIP; bottom panel) antibody. Wild, wild type; Hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: In Vivo, Labeling, Immunoprecipitation, Kinase Assay, Expressing, Western Blot

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on downstream kinases from PI 3-kinase. (a) IGF-1-induced Akt activity in cells of each genotype. Cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 5 min. Cell lysates were subjected to immunoblotting with anti-phospho-Akt (αphospho-Akt; top panel) antibody or immunoprecipitation with αAkt antibody. The immunoprecipitates were subjected to an immune complex kinase assay. In the bottom panel, each bar represents the mean ± standard deviation of the relative Akt kinase activity calculated from the results of three independent experiments. *, P value of <0.01 for wild-type (Wild) versus heterozygous KO (Hetero) cells; **, P value of <0.05 for wild versus null cells. (b) IGF-1-induced p70S6K activity in cells of each genotype. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoblotting with anti-phospho-p70S6K (αphospho-p70S6K; top panel) antibody or immunoprecipitation with αp70S6K antibody. The immunoprecipitates were subjected to an immune complex kinase assay. In the bottom panel, each bar represents the mean ± standard deviation of the relative p70S6K kinase activity calculated from the results of three independent experiments.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Immune Complex Kinase Assay, Standard Deviation

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on serum deprivation-induced apoptosis and IGF-1-dependent antiapoptosis. Cells were cultured in the indicated concentration of serum (left panel) or IGF-1 without serum (right panel) for 5 h. The level of apoptosis was assessed using an enzyme-linked immunosorbent assay for nucleosomal DNA as described in Materials and Methods. Each value is expressed as the ratio of the value of the wild-type cells treated with 10% serum and represents the mean ± standard deviation of three independent experiments. Wild, wild type; Hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on IGF-1-dependent antiapoptotic signaling. (a) IGF-1-induced Bad phosphorylation and the interaction between Bad and 14-3-3 in cells of each genotype. Cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 20 min. Cell lysates were subjected to immunoprecipitation with αBad antibody followed by immunoblotting. Immunoblots were probed with αBad (top panel), anti-phospho-Bad (αphospho-Bad; middle panel), or anti-14-3-3 (α14-3-3; bottom panel) antibody and visualized by enhanced chemiluminescence with protein A-conjugated peroxidase. (b) IGF-1-induced p90RSK activity in cells of each genotype. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoprecipitation with αp90RSK antibody followed by an immune complex kinase assay. Each bar represents the mean ± standard deviation of the p90RSK activity calculated from the results of three independent experiments. *, P value of <0.01 for wild-type (Wild) versus null cells. (c) IGF-1-induced FKHR phosphorylation. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoblotting with anti-phospho-FKHR (αphospho-FKHR; top panel) or anti-FKHR (αFKHR; bottom panel) antibody. (d) IGF-1-induced CREB phosphorylation in cells of each genotype. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoblotting with anti-phospho-CREB (αphospho-CREB; top panel) or anti-CREB (αCREB; bottom panel) antibody. Hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Immune Complex Kinase Assay, Standard Deviation