Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

Article Snippet: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

Techniques: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

Article Snippet: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

Techniques: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

Journal: Cell reports

Article Title: The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

doi: 10.1016/j.celrep.2022.110789

Figure Lengend Snippet: Figure 1. Two-step Cdk1 inactivation during fertilization (A) Representative time-lapse images of spindles and chromosomes in live MII oocytes undergoing fertilization. White arrow, fertilization cone and sperm DNA. Note that the anaphase II spindle with condensed chromosomes at the poles persists for over 2 h (n = 12). Time is hh:mm post-anaphase onset. Scale bar, 10 mm (see Video S1). (B) FRET ratios of a biosensor of Cdk1 activity normalized to 100% at the time of insemination and sizes of the H2B-RFP signals for male and female DNA in live MII oocytes during fertilization (n = 15). FRET ratio at the time of insemination ranged from 0.78 to 0.81. Blue arrow highlights time of anaphase II. Data are mean ± SEM. See also Figures S1A and S1B. (C) Representative images of fertilized MII oocytes immunostained for tubulin, DNA, and the nuclear envelope marker lamin (n > 15). Yellow arrowhead, faint lamin staining. Time is hh:mm post-insemination. Results are representative of at least three independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Hira Active motif Cat#39558;RRID: AB_2793256 Anti-Cyclin B1 Abcam Cat#ab72; RRID: AB_305751 Anti-p-Cdk1 Cell Signaling Cat#9111S;RRID: AB_331460 Anti-CDK1 Cell Signaling Cat#77055;RRID: AB_2716331 Anti-Vinculin Sigma Aldrich Cat#V9131;RRID: AB_477629 Rabbit anti-b-tubulin Sigma-Aldrich Cat# SAB2700070 HRP-conjugated goat anti-rabbit Bio-Rad Cat#1706515;RRID: AB_11125142 HRP-conjugated goat anti-mouse Bio-Rad Cat#1706516;RRID: AB_11125547 Anti-Cep55 Kalimutho et al. (2018) Dr. Kum K. Khanna Anti-Wee1B Oh et al. (2011) Dr. Marco Conti Anti-Lamin Abcam Cat#ab108922;RRID: AB_10860619 Hoechst 33342 Sigma Cat#14533 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular Probes Cat#A11029;RRID: AB_2534088 Alexa Fluor 488 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11034;RRID: AB_142134 Alexa Fluor 568 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11011;RRID: AB_143157 Alexa Fluor 633 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A21070;RRID: AB_2535731 Alexa Fluor 633 goat anti-mouse IgG (H+L) Molecular Probes Cat#A21052;RRID: AB_2535719 Bacterial and virus strains Cdk1 FRET Sensor Addgene Addgene_26064;RRID: Addgene_26064 Chemicals, peptides, and recombinant proteins 3-Isobutyl-1-methylxanthine (IBMX) Sigma-Aldrich Cat#I7018 PMS (pregnant mare’s serum) MSD Folligon 1000 I.E. hCG (human chorionic gonadotropin) MSD Chorulon 1500 I.E.

Techniques: Activity Assay, Marker, Staining

Journal: Cell reports

Article Title: The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

doi: 10.1016/j.celrep.2022.110789

Figure Lengend Snippet: Figure 2. Post-anaphase Cdk1 activity is important for male PN formation (A) Representative time-lapse images of spindles and chromosomes in inseminated MII oocytes treated with either flavopiridol (n = 14) or DMSO (n = 14) after anaphase II. Time is hh:mm post-anaphase onset. Scale bar, 10 mm (see Video S2). Note that both male and female DNA undergo marked decondensation in DMSO-treated oocytes, whereas following flavopiridol treatment, only female DNA undergoes decondensation. (B) FRET ratios of a biosensor of Cdk1 activity (n = 11) normalized to 100% at the time of insemination and sizes of the H2B-RFP signals for male (n = 14) and female (n = 15) DNA in live MII oocytes treated with flavopiridol after anaphase II (n = 15). FRET ratios at the time of insemination ranged from 0.71 to 0.81 (n = 11). Data are mean ± SEM. See also Figures S1A and S1C. (C) Shown are representative images of fertilized MII oocytes immunostained for Hira and DNA following treatment with either flavopiridol or DMSO after anaphase II. Note the intense Hira staining within the male DNA following DMSO but not flavopiridol treatment (n > 20 per group). Scale bar, 20 mm. (D and E) Samples (50 oocytes per lane) of flavopiridol- and DMSO-treated MII oocytes were immunoblotted for Hira and actin at 6 h post-insemination. Shown is a representative immunoblot (D) and quantification of band intensities normalized to values in DMSO-treated controls (E). Data are mean ± SEM; ns, p > 0.05 (Student’s t test). Results are representative of at least three independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Hira Active motif Cat#39558;RRID: AB_2793256 Anti-Cyclin B1 Abcam Cat#ab72; RRID: AB_305751 Anti-p-Cdk1 Cell Signaling Cat#9111S;RRID: AB_331460 Anti-CDK1 Cell Signaling Cat#77055;RRID: AB_2716331 Anti-Vinculin Sigma Aldrich Cat#V9131;RRID: AB_477629 Rabbit anti-b-tubulin Sigma-Aldrich Cat# SAB2700070 HRP-conjugated goat anti-rabbit Bio-Rad Cat#1706515;RRID: AB_11125142 HRP-conjugated goat anti-mouse Bio-Rad Cat#1706516;RRID: AB_11125547 Anti-Cep55 Kalimutho et al. (2018) Dr. Kum K. Khanna Anti-Wee1B Oh et al. (2011) Dr. Marco Conti Anti-Lamin Abcam Cat#ab108922;RRID: AB_10860619 Hoechst 33342 Sigma Cat#14533 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular Probes Cat#A11029;RRID: AB_2534088 Alexa Fluor 488 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11034;RRID: AB_142134 Alexa Fluor 568 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11011;RRID: AB_143157 Alexa Fluor 633 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A21070;RRID: AB_2535731 Alexa Fluor 633 goat anti-mouse IgG (H+L) Molecular Probes Cat#A21052;RRID: AB_2535719 Bacterial and virus strains Cdk1 FRET Sensor Addgene Addgene_26064;RRID: Addgene_26064 Chemicals, peptides, and recombinant proteins 3-Isobutyl-1-methylxanthine (IBMX) Sigma-Aldrich Cat#I7018 PMS (pregnant mare’s serum) MSD Folligon 1000 I.E. hCG (human chorionic gonadotropin) MSD Chorulon 1500 I.E.

Techniques: Activity Assay, Staining, Western Blot

Journal: Cell reports

Article Title: The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

doi: 10.1016/j.celrep.2022.110789

Figure Lengend Snippet: Figure 3. Premature Cdk1 inactivation is detrimental to embryo development (A and B) Representative time-lapse images of spindles and chromosomes during the first embryonic division following treatment of MII oocytes with either DMSO (n = 12) or flavopiridol (n = 11) from 3 to 6 h post-insemination. Time is hh:mm relative to anaphase onset. Scale bar, 10 mm. See Videos S3 and S4. Yellow arrow (B) highlights lagging chromosomes. (C) Embryonic development rates following treatment of MII oocytes from 3 to 6 h post-insemination with either DMSO (n = 30) or flavopiridol (n = 35) and following treatment with flavopiridol from 7 to 10 h post-insemination (n = 40). Data in graph are mean ± SEM; ***p < 0.001, 2-tailed Student’s t test. Scale bar, 10 mm. Results are representative of at least three independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Hira Active motif Cat#39558;RRID: AB_2793256 Anti-Cyclin B1 Abcam Cat#ab72; RRID: AB_305751 Anti-p-Cdk1 Cell Signaling Cat#9111S;RRID: AB_331460 Anti-CDK1 Cell Signaling Cat#77055;RRID: AB_2716331 Anti-Vinculin Sigma Aldrich Cat#V9131;RRID: AB_477629 Rabbit anti-b-tubulin Sigma-Aldrich Cat# SAB2700070 HRP-conjugated goat anti-rabbit Bio-Rad Cat#1706515;RRID: AB_11125142 HRP-conjugated goat anti-mouse Bio-Rad Cat#1706516;RRID: AB_11125547 Anti-Cep55 Kalimutho et al. (2018) Dr. Kum K. Khanna Anti-Wee1B Oh et al. (2011) Dr. Marco Conti Anti-Lamin Abcam Cat#ab108922;RRID: AB_10860619 Hoechst 33342 Sigma Cat#14533 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular Probes Cat#A11029;RRID: AB_2534088 Alexa Fluor 488 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11034;RRID: AB_142134 Alexa Fluor 568 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11011;RRID: AB_143157 Alexa Fluor 633 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A21070;RRID: AB_2535731 Alexa Fluor 633 goat anti-mouse IgG (H+L) Molecular Probes Cat#A21052;RRID: AB_2535719 Bacterial and virus strains Cdk1 FRET Sensor Addgene Addgene_26064;RRID: Addgene_26064 Chemicals, peptides, and recombinant proteins 3-Isobutyl-1-methylxanthine (IBMX) Sigma-Aldrich Cat#I7018 PMS (pregnant mare’s serum) MSD Folligon 1000 I.E. hCG (human chorionic gonadotropin) MSD Chorulon 1500 I.E.

Techniques:

Journal: Cell reports

Article Title: The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

doi: 10.1016/j.celrep.2022.110789

Figure Lengend Snippet: Figure 4. Wee1B mediates both phases of Cdk1 inactivation (A and B) Samples of MII oocytes (n = 50) were immunoblotted for cyclin B1, Tyr15-phosphory- lated Cdk1 (p-Cdk1), total Cdk1, and vinculin (loading control) at 3 and 6 h post-insemination. Shown is a representative immunoblot (A) and quantification of band intensities normalized to values at 6 h post-insemination (B) Data are mean ± SEM. ***p < 0.001; ns, p > 0.05 (Student’s t test). See also Figures S2D and S2E. (C) FRET ratios of a Cdk1 biosensor normalized to 100% at the time of insemination (n = 11) and sizes of the H2B-RFP signals for male and female DNA following treatment with the Wee1B inhibitor MK1775 at the time of insemination (pre-anaphase onset). FRET ratios at the time of insemination ranged from 0.81 to 0.82 (n = 11). Data are mean ± SEM. (D) Representative time-lapse images of spindles andchromosomes(n=20)foranoocytetreatedasin (C). Time is hh:mm post-insemination. Note the absence of anaphase. White arrow indicates sperm DNA. Scale bar, 10 mm. See also Figures S2A–S2C. (E) Normalized Cdk1 activity (n = 22) and sizes of the H2B-RFP signals for male and female DNA in inseminated MII oocytes treated with MK1775 at 2 h post-insemination (post-anaphase). Data are mean ± SEM. (F) Representative time-lapse images of spindles andchromosomes(n=15)foranoocytetreatedasin (E). White arrow indicates sperm DNA. Time is hh:mm post-insemination. Results are representa- tiveof at least three independent experiments. Scale bar, 10 mm. See also Figures S2A–S2C.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Hira Active motif Cat#39558;RRID: AB_2793256 Anti-Cyclin B1 Abcam Cat#ab72; RRID: AB_305751 Anti-p-Cdk1 Cell Signaling Cat#9111S;RRID: AB_331460 Anti-CDK1 Cell Signaling Cat#77055;RRID: AB_2716331 Anti-Vinculin Sigma Aldrich Cat#V9131;RRID: AB_477629 Rabbit anti-b-tubulin Sigma-Aldrich Cat# SAB2700070 HRP-conjugated goat anti-rabbit Bio-Rad Cat#1706515;RRID: AB_11125142 HRP-conjugated goat anti-mouse Bio-Rad Cat#1706516;RRID: AB_11125547 Anti-Cep55 Kalimutho et al. (2018) Dr. Kum K. Khanna Anti-Wee1B Oh et al. (2011) Dr. Marco Conti Anti-Lamin Abcam Cat#ab108922;RRID: AB_10860619 Hoechst 33342 Sigma Cat#14533 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular Probes Cat#A11029;RRID: AB_2534088 Alexa Fluor 488 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11034;RRID: AB_142134 Alexa Fluor 568 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11011;RRID: AB_143157 Alexa Fluor 633 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A21070;RRID: AB_2535731 Alexa Fluor 633 goat anti-mouse IgG (H+L) Molecular Probes Cat#A21052;RRID: AB_2535719 Bacterial and virus strains Cdk1 FRET Sensor Addgene Addgene_26064;RRID: Addgene_26064 Chemicals, peptides, and recombinant proteins 3-Isobutyl-1-methylxanthine (IBMX) Sigma-Aldrich Cat#I7018 PMS (pregnant mare’s serum) MSD Folligon 1000 I.E. hCG (human chorionic gonadotropin) MSD Chorulon 1500 I.E.

Techniques: Control, Western Blot, Activity Assay

Journal: Cell reports

Article Title: The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

doi: 10.1016/j.celrep.2022.110789

Figure Lengend Snippet: Figure 5. Cep55 regulates Cdk1 inactivation (A) Shown are representative images of MII oocytes immunostained for Cep55 at the times shown post-insemination. Note that, around the time of the second phase of Cdk1 inactivation (5 h post-insemination), Cep55 exhibits strong localization to the spindle midzone and midzone constriction (white arrow). Scale bar, 10 mm. See also Figure S5A. (B) FRET ratios of a Cdk1 biosensor normalized to 100% at the time of insemination and sizes of the H2B-RFP signals for male and female DNA in mock-depleted (n = 13), Cep55-depleted (n = 13), and Cep55-overexpressing MII oocytes (n = 11). FRET ratios at the time of insemination ranged from 0.77 to 0.82. Data are mean ± SEM. See also Figures S4A–S4G. (C) Representative time-lapse images of spindles and chromosomes in mock-depleted (n = 20), Cep55-depleted (n = 20), and Cep55-overexpressing (n = 18) MII oocytes during fertilization. Note that, due to the lack of decondensation, male DNA is not readily visible in Cep55-overexpressing oocytes (+Cep55 mRNA). Time is hh:mm post-anaphase onset. Scale bar, 15 mm. Results are representative of at least three independent experiments. See also Figure S5B.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Hira Active motif Cat#39558;RRID: AB_2793256 Anti-Cyclin B1 Abcam Cat#ab72; RRID: AB_305751 Anti-p-Cdk1 Cell Signaling Cat#9111S;RRID: AB_331460 Anti-CDK1 Cell Signaling Cat#77055;RRID: AB_2716331 Anti-Vinculin Sigma Aldrich Cat#V9131;RRID: AB_477629 Rabbit anti-b-tubulin Sigma-Aldrich Cat# SAB2700070 HRP-conjugated goat anti-rabbit Bio-Rad Cat#1706515;RRID: AB_11125142 HRP-conjugated goat anti-mouse Bio-Rad Cat#1706516;RRID: AB_11125547 Anti-Cep55 Kalimutho et al. (2018) Dr. Kum K. Khanna Anti-Wee1B Oh et al. (2011) Dr. Marco Conti Anti-Lamin Abcam Cat#ab108922;RRID: AB_10860619 Hoechst 33342 Sigma Cat#14533 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular Probes Cat#A11029;RRID: AB_2534088 Alexa Fluor 488 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11034;RRID: AB_142134 Alexa Fluor 568 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11011;RRID: AB_143157 Alexa Fluor 633 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A21070;RRID: AB_2535731 Alexa Fluor 633 goat anti-mouse IgG (H+L) Molecular Probes Cat#A21052;RRID: AB_2535719 Bacterial and virus strains Cdk1 FRET Sensor Addgene Addgene_26064;RRID: Addgene_26064 Chemicals, peptides, and recombinant proteins 3-Isobutyl-1-methylxanthine (IBMX) Sigma-Aldrich Cat#I7018 PMS (pregnant mare’s serum) MSD Folligon 1000 I.E. hCG (human chorionic gonadotropin) MSD Chorulon 1500 I.E.

Techniques:

Journal: Cell reports

Article Title: The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

doi: 10.1016/j.celrep.2022.110789

Figure Lengend Snippet: Figure 6. Nocodazole causes premature Cdk1 inactivation (A and B) Shown are representative images of MII oocytes immunostained for Wee1B and tubulin following treatment with either DMSO (n = 15) or nocodazole (n = 15) at 2 h post-insemination (post-anaphase) (A) and quantification of the fluorescence intensity of the Wee1B signal in the cytoplasm (B). Data are mean ± SEM. ***p < 0.001 (Student’s t test). Scale bar, 10 mm. See also Figures S3A–S3F. (C) Representative time-lapse images of spindles and chromosomes in MII oocytes treated with either DMSO (n = 20) or nocodazole (n = 15) at 2 h post- insemination. Note that both male (white arrow) and female (yellow arrow) DNA undergo decondensation in DMSO-treated oocytes, whereas only female DNA can be seen to decondense following nocodazole treatment. Time is hh:mm post-anaphase-onset. Scale bar, 10 mm. (D and E) FRET ratios of a Cdk1 biosensor normalized to 100% at the time of insemination and sizes of the H2B-RFP signals for male and female DNA following treatment with either DMSO (n = 13, D) or nocodazole (n = 11, E) at 2 h post-insemination. FRET ratios at the time of insemination ranged from 0.77 to 0.81. Data are mean ± SEM. Results are representative of at least three independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Hira Active motif Cat#39558;RRID: AB_2793256 Anti-Cyclin B1 Abcam Cat#ab72; RRID: AB_305751 Anti-p-Cdk1 Cell Signaling Cat#9111S;RRID: AB_331460 Anti-CDK1 Cell Signaling Cat#77055;RRID: AB_2716331 Anti-Vinculin Sigma Aldrich Cat#V9131;RRID: AB_477629 Rabbit anti-b-tubulin Sigma-Aldrich Cat# SAB2700070 HRP-conjugated goat anti-rabbit Bio-Rad Cat#1706515;RRID: AB_11125142 HRP-conjugated goat anti-mouse Bio-Rad Cat#1706516;RRID: AB_11125547 Anti-Cep55 Kalimutho et al. (2018) Dr. Kum K. Khanna Anti-Wee1B Oh et al. (2011) Dr. Marco Conti Anti-Lamin Abcam Cat#ab108922;RRID: AB_10860619 Hoechst 33342 Sigma Cat#14533 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular Probes Cat#A11029;RRID: AB_2534088 Alexa Fluor 488 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11034;RRID: AB_142134 Alexa Fluor 568 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A11011;RRID: AB_143157 Alexa Fluor 633 goat anti-rabbit IgG (H+L) Molecular Probes Cat#A21070;RRID: AB_2535731 Alexa Fluor 633 goat anti-mouse IgG (H+L) Molecular Probes Cat#A21052;RRID: AB_2535719 Bacterial and virus strains Cdk1 FRET Sensor Addgene Addgene_26064;RRID: Addgene_26064 Chemicals, peptides, and recombinant proteins 3-Isobutyl-1-methylxanthine (IBMX) Sigma-Aldrich Cat#I7018 PMS (pregnant mare’s serum) MSD Folligon 1000 I.E. hCG (human chorionic gonadotropin) MSD Chorulon 1500 I.E.

Techniques:

Journal: American Journal of Translational Research

Article Title: S-phase kinase-associated protein 2 impairs the inhibitory effects of miR-1236-3p on bladder tumors

doi:

Figure Lengend Snippet: Primers used in this study

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA) and then incubated with primary antibodies, including those against Skp2 (1/1000) (Affinity, USA), P21 (1/1000) (BD Biosciences), P27 (1/1000) (Affinity, USA), CDK1 (1/1000) (Boster, China), GAPDH (1/500) (Boster, China) and β-actin (1/500) (Boster, China), overnight at 4°C.

Techniques: Control

Journal: American Journal of Translational Research

Article Title: S-phase kinase-associated protein 2 impairs the inhibitory effects of miR-1236-3p on bladder tumors

doi:

Figure Lengend Snippet: miR-1236 induced Skp2 expression independent of p21 activation and influenced downstream Skp2 gene expression. A. Skp2 mRNA levels were evaluated by qRT-PCR. **P<0.01 compared to control miRNA group. B. Expression of Skp2 protein was further detected by Western blot analysis. Only miR-1236 up-regulated Skp2 expression. C. Expression of p21 and Skp2 mRNA in BCa cells was detected by qRT-PCR. *P<0.05, **P<0.01 compared to control miRNA group. D. Western blotting was conducted to detect the expression of p21 and Skp2 protein in BCa cells. Knockdown with sip21 had no influence on the up-regulation of Skp2 by miR-1236. E. mRNA expression levels of p27 and CDK1 were assessed by qRT-PCR. *P<0.05 compared to the control miRNA group. F. P27 and CDK1 expression levels were detected by Western blot in 5637 and T24 cells. miR-1236 decreased p27 expression and up-regulated CDK1 expression. dsRNA-245 had no significant effect on p27 and CDK1. G. Expression of Skp2, p27 and CDK1 was detected by qRT-PCR. GAPDH served as an internal control. **P<0.01, ***P<0.001 compared with the miR-1236 group. H. Skp2, p27 and CDK1 protein expression levels were detected by Western blot. β-actin levels were used as an internal control. miR-1236 influenced p27 and CDK1 expression through up-regulation of Skp2.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA) and then incubated with primary antibodies, including those against Skp2 (1/1000) (Affinity, USA), P21 (1/1000) (BD Biosciences), P27 (1/1000) (Affinity, USA), CDK1 (1/1000) (Boster, China), GAPDH (1/500) (Boster, China) and β-actin (1/500) (Boster, China), overnight at 4°C.

Techniques: Expressing, Activation Assay, Gene Expression, Quantitative RT-PCR, Control, Western Blot, Knockdown