2 ap rabbit polyclonal anti notch2 proteintech  (Proteintech)

Journal: eLife

Article Title: SMAD4 promotes somatic-germline contact during murine oocyte growth

doi: 10.7554/elife.91798

Figure Lengend Snippet: Figure 1. SMAD4 is efficiently depleted in granulosa cells of granulosa cell-oocyte complexes (GOCs) in vitro using tamoxifen-induced Cre recombinase. (A) Immunoblot of primordial follicles, growing follicles, and granulosa cells and oocytes isolated from antral follicles, probed using anti-SMAD4 and anti-SMAD2/3. (B) Breeding scheme to delete Smad4 in the granulosa cells using Amhr2-Cre. The mTmG cassette enables Cre recombinase activity to be verified through expression of EGFP. (C) Left: Confocal images of histological sections of paraffin-embedded 12-d ovary (i,

Article Snippet: J. Bernard, McGill University strain, strain background (M. musculus) Amhr2tm3(cre)Bhr MMRRC strain 014245- UNC Supplied by Dr. Makoto Nagano, McGill University strain, strain background (M. musculus) B6.129Gt(ROSA)26Sortm1(cre/ ERT2)Tyj/J Jackson Laboratories strain 008463 strain, strain background (M. musculus) B6.129(Cg)Gt(ROSA)26Sortm4(ACTBtdTomato,-EGFP)Luo/J Jackson Laboratories strain 007676 antibody anti- SMAD2/3 (Rabbit monoclonal) Cell Signaling Technology 8685 RRID: AB_10889933 1:1000 (immunoblot) antibody anti- SMAD4 (Rabbit polyclonal) Sigma HPA019154 RRID: AB_1853480 1:1000 (immunoblot) antibody anti- N- cadherin (Mouse monoclonal) BD Biosciences B610920 RRID: AB_2077527 1:1000 (immunoblot) antibody anti- Notch2 (Rabbit polyclonal) Abcam Ab8926 RRID: AB_2267338 1:1000 (immunoblot) antibody anti- GFP (Rabbit polyclonal) Invitrogen A11122 RRID: AB_2307355 1:100 (immunofluorescence, immunohistochemistry) Granados- Aparici et al. eLife 2024;13:RP91798.

Techniques: In Vitro, Western Blot, Isolation, Activity Assay, Expressing

Journal: Cell Death and Differentiation

Article Title: Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation

doi: 10.1038/s41418-021-00861-5

Figure Lengend Snippet: A Immunoprecipitation with control IgG and Gm364 antibody was performed and followed by SDS-PAGE and silver staining. Then distinct bands were sent for MALDI. TTC37, MIB2, and GRAMD1A were identified as Gm364-interacting proteins. B RT-PCR showed that within oocytes, Notch2 is the most abundant among Notch family members 1–4. C Immunofluorescence showed that Notch2 was enriched on the oocyte membrane. DNA in blue, Notch2 in green. D Western blot showed that Notch2 is more abundant in oocytes than in granular cells. E Co-IP and blots showed that Gm364 interacts with Notch2 in oocytes. F . Western blot showed that NICD2 was more abundant in oocytes than in granular cells. G Blot showed that NICD2 protein levels decreased gradually during oocyte meiosis. H – J Immunofluorescence and blot showed that Gm364 knockout significantly decreased the NICD2 protein level. DNA in blue, NICD2 in green. K Blot showed that γ-secretase inhibition significantly decreased NICD2 levels. L. NICD2 reduction by γ-secretase inhibition greatly decreased the percentage of MII oocytes. M , N Immunofluorescence of in vitro fertilized oocytes and quantification showed that inhibiting γ-secretase significantly decreased the percentage of fertilized oocytes and the percentage of 2-PN (two pronucleus). PNs in the control oocyte or chromosomes in the γ-secretase-inhibited (γ-secretase(-)) oocytes were delineated with red dot-line circle; polar bodies (pbs) were labeled with arrows. DNA in blue, tubulin in green. β-actin or α-tubulin was used as a loading control. Scale bar, 20 μm. *Indicates p < 0.05.

Article Snippet: Primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti-β-Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti-β-Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti‐alpha Tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss, Beijing, China); Human anti-centromere CREST antibody (cat#: 15-234; Antibodies Incorporated, USA); rabbit polyclonal anti-Notch2 (cat#: ab8926, Abcam, Cambridge, UK); Rabbit polyclonal anti-cleaved Notch2 (Asp 1733) antibody (cat#:AF5255; Affinity biosciences, OH, USA); Anti-DLL3 rabbit polyclonal antibody (cat#: Ab103102; Abcam, Cambridge, UK); Anti-MIB2 rabbit polyclonal antibody (cat#: A17829; ABclonal, MA, USA); anti-Phosphorylation RICTOR (Thr1135) (cat#: D30A3, Cell Signaling Technology, MA, USA); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phospho AKT (Thr308, Cat#: 13038, Cell Signaling Technology); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science).

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Membrane, Western Blot, Co-Immunoprecipitation Assay, Knock-Out, Inhibition, In Vitro, Labeling

Journal: Cell Death and Differentiation

Article Title: Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation

doi: 10.1038/s41418-021-00861-5

Figure Lengend Snippet: A , B RT-PCR and quantification showed that Mib2 mRNA is more abundant than Mib1 mRNA within oocytes. C , D Blots and quantification showed that MIB2 is more abundant in oocytes than in granular cells. E Co-IP and blot showed that Gm364 interacts with MIB2. F Co-IP and blot showed that Notch2 interacts with MIB2. G , H Blots and quantification showed that Gm364 knockout did not affect MIB2 protein levels. I , J Immunofluorescence and quantification showed that MIB2 was enriched on the membrane in WT oocytes, whereas the membrane enrichment completely disappeared and cytoplasmic MIB2 increased in Gm364 knockout oocytes. K , L RT-PCR showed that among Dll members, Dll3 was the most abundant in oocytes. M , N Blots and quantification showed that DLL3 is more abundant in oocytes (oo) than in granular cells (GCs). O Co-IP and blot showed that Gm364 interacts with DLL3. P Co-IP and blot showed that Notch2 interacts with DLL3. Q , R DLL3 antibody IP and Ub46 blots showed that Gm364 knockout significantly decreased DLL3 ubiquitination levels. S , T Blot and quantification showed that Gm364 knockout significantly decreased DLL3 protein levels. U Immuno-EM showed that MIB2 and DLL3 localized at the oocyte membrane and were close (<20 nm) to each other. MIB2 and DLL3 primary antibodies were bound with 15-nm and 35-nm gold-conjugated secondary antibodies, respectively. V Immuno-EM showed that MIB2 and Gm364 localized at the oocyte membrane and were close (<20 nm) to each other. MIB2 and Gm364 primary antibodies were bound with 10-nm and 15-nm gold-conjugated secondary antibodies, respectively. β-Actin or GAPDH was used as a loading control. Scale bar in panel ( I ), 20 μm; scale bar in panels ( U ) and ( V ), 50 nm. *Indicates p < 0.05.

Article Snippet: Primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti-β-Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti-β-Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti‐alpha Tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss, Beijing, China); Human anti-centromere CREST antibody (cat#: 15-234; Antibodies Incorporated, USA); rabbit polyclonal anti-Notch2 (cat#: ab8926, Abcam, Cambridge, UK); Rabbit polyclonal anti-cleaved Notch2 (Asp 1733) antibody (cat#:AF5255; Affinity biosciences, OH, USA); Anti-DLL3 rabbit polyclonal antibody (cat#: Ab103102; Abcam, Cambridge, UK); Anti-MIB2 rabbit polyclonal antibody (cat#: A17829; ABclonal, MA, USA); anti-Phosphorylation RICTOR (Thr1135) (cat#: D30A3, Cell Signaling Technology, MA, USA); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phospho AKT (Thr308, Cat#: 13038, Cell Signaling Technology); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science).

Techniques: Reverse Transcription Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Knock-Out, Immunofluorescence, Membrane

Journal: Cell Death and Differentiation

Article Title: Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation

doi: 10.1038/s41418-021-00861-5

Figure Lengend Snippet: A , G , J , Q To investigate the signal pathway of how Gm364 regulates AKT activation, we created four working models and examined which was the correct pathway. The upstream parts for all four models, i.e., Gm364 binds with MIB2 and DLL3 to promote the cleavage of Notch2 into active NICD2, were the same, while the signals downstream from NICD2 were distinct from each other. A – F Model 1: if NICD2 first activates mTORC2, then mTORC2 activates AKT, we expected that Gm364 knockout would significantly decrease p-RICTOR (a component of mTORC2). However, p-RICTOR was unaltered by Gm364 knockout ( B ) and ( C ), and p-RICTOR did not interact with Gm364 ( D ), Notch2 ( E ), and NICD2 ( F ), suggesting that Model 1 was incorrect. G – I Model 2: If NICD2 entered the nucleus to promote Akt transcription, we expected that Gm364 knockout would significantly decrease Akt mRNA levels. However, Akt mRNA was unaltered, suggesting that Model 2 was also incorrect. J – P Model 3: if NICD2 entered the nucleus to inhibit Pten transcription, whereas PTEN negatively regulated AKT, we expected that Gm364 knockout would significantly increase PTEN protein and mRNA levels. However, neither PTEN protein ( K ) and ( L ) nor mRNA level ( M ) and ( N ) was altered, and neither NICD2 ( O ) nor Gm364 ( P ) interact with PTEN, suggesting that Model 3 was also incorrect. Q – U Model 4: if NICD2 could directly activate (phosphorylate) AKT, we expected that Gm364 knockout would significantly decrease p-AKT. Here, Gm364 knockout significantly reduced AKT phosphorylation at both S473 and T308 ( R ) and ( S ), and both NICD2 ( T ) and Gm364 interact with AKT ( U ), suggesting that Model 4 was correct. GAPDH was used as a loading control.

Article Snippet: Primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti-β-Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti-β-Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti‐alpha Tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss, Beijing, China); Human anti-centromere CREST antibody (cat#: 15-234; Antibodies Incorporated, USA); rabbit polyclonal anti-Notch2 (cat#: ab8926, Abcam, Cambridge, UK); Rabbit polyclonal anti-cleaved Notch2 (Asp 1733) antibody (cat#:AF5255; Affinity biosciences, OH, USA); Anti-DLL3 rabbit polyclonal antibody (cat#: Ab103102; Abcam, Cambridge, UK); Anti-MIB2 rabbit polyclonal antibody (cat#: A17829; ABclonal, MA, USA); anti-Phosphorylation RICTOR (Thr1135) (cat#: D30A3, Cell Signaling Technology, MA, USA); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phospho AKT (Thr308, Cat#: 13038, Cell Signaling Technology); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science).

Techniques: Activation Assay, Knock-Out

Journal: Cell Death and Differentiation

Article Title: Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation

doi: 10.1038/s41418-021-00861-5

Figure Lengend Snippet: On the oocyte membrane, Notch2 and its interactors might transduce signals from outside into the cytoplasm. Gm364 might be a novel essential component of the Notch-containing upstream signal launcher. Gm364 is essential for the membrane enrichment of MIB2, while MIB2 is a ubiquitin ligase required for DLL3 ubiquitination. Next, ubiquitinated DLL3, the active form of DLL3, binds and activates Notch2. Then, the activated Notch2 is cleaved within the cytoplasm to produce NICD2, the intracellular active domain of Notch2. Finally, NICD2 can directly activate AKT within the cytoplasm to regulate oocyte meiosis and quality.

Article Snippet: Primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti-β-Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti-β-Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti‐alpha Tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss, Beijing, China); Human anti-centromere CREST antibody (cat#: 15-234; Antibodies Incorporated, USA); rabbit polyclonal anti-Notch2 (cat#: ab8926, Abcam, Cambridge, UK); Rabbit polyclonal anti-cleaved Notch2 (Asp 1733) antibody (cat#:AF5255; Affinity biosciences, OH, USA); Anti-DLL3 rabbit polyclonal antibody (cat#: Ab103102; Abcam, Cambridge, UK); Anti-MIB2 rabbit polyclonal antibody (cat#: A17829; ABclonal, MA, USA); anti-Phosphorylation RICTOR (Thr1135) (cat#: D30A3, Cell Signaling Technology, MA, USA); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phospho AKT (Thr308, Cat#: 13038, Cell Signaling Technology); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science).

Techniques: Membrane