mouse  (Santa Cruz Biotechnology)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Nuclear translocation of ISG15 regulated by PPP2R2B inhibits cisplatin resistance of bladder cancer

doi: 10.1007/s00018-024-05320-1

Figure Lengend Snippet: The top 5 proteins identified by mass spectrometry after Co-IP

Article Snippet: Antibodies used for western blotting were as follows: PPP2R2B (ab157461, Abcam), GAPDH (60004-1-Ig, Proteintech), beta tubulin (10068-1-AP, Proteintech), phospho-DNA PKcs (Ser2056) (ab124918, Abcam), phospho-ATR (Ser428) (2853, CST), phospho-ATM (Ser1981) (5883, CST), ISG15 (15981-1-AP, Proteintech), anti-phosphoserine/threonine (PP2551, ECMbio), IPO5 (sc-55,527, Santa Cruz), LaminA/C (10298-1-AP, Proteintech), Human-Reactive STING Pathway Antibody Sampler Kit (38,866, CST), FLAG (F1804, Sigma), HA (51064-2-AP, Proteintech), STAT1 (9172, CST), pSTAT1 (9167, CST), H3 (17168-1-AP, Proteintech), H3K9me3 (ab8898, Abcam), and SUV39H1 (05-615, Sigma).

Techniques: Mass Spectrometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Nuclear translocation of ISG15 regulated by PPP2R2B inhibits cisplatin resistance of bladder cancer

doi: 10.1007/s00018-024-05320-1

Figure Lengend Snippet: PPP2R2B facilitates binding of IPO5 and ISG15 and transports ISG15 into the nucleus. ( A ) KEGG enrichment analysis of proteins that interacted with PPP2R2B in BC. Nucleocytoplasmic transport was the top label. ( B ) Western blot to detect the interaction between PPP2R2B and IPO5/ISG15 after Co-IP of PPP2R2B. ( C ) Western blot to detected the interaction between IPO5 and PPP2R2B/ISG15 after Co-IP of IPO5. PPP2R2B overexpression enhanced the binding between IPO5 and ISG15. ( D ) Colocalization of PPP2R2B, IPO5, and ISG15 in J82 and T24 cells, demonstrated by immunofluorescence. ( E ) Subcellular localization of ISG15 in PPP2R2B-silenced J82 cells and PPP2R2B-overexpressing T24 cells demonstrated by immunofluorescence. ( F ) Illustration of the NLS-defective PPP2R2B construct (2R2BΔNLS). ( G ) Subcellular localization of ISG15 in control, PPP2R2B and 2R2BΔNLS overexpression group. ( H ) PPP2R2B and ISG15 RNA levels in control, PPP2R2B and 2R2BΔNLS overexpression group. ( n = 3; one-way ANOVA with Dunnett’s test). Three independent experiments were performed. Error bars were represented as mean ± SD

Article Snippet: Antibodies used for western blotting were as follows: PPP2R2B (ab157461, Abcam), GAPDH (60004-1-Ig, Proteintech), beta tubulin (10068-1-AP, Proteintech), phospho-DNA PKcs (Ser2056) (ab124918, Abcam), phospho-ATR (Ser428) (2853, CST), phospho-ATM (Ser1981) (5883, CST), ISG15 (15981-1-AP, Proteintech), anti-phosphoserine/threonine (PP2551, ECMbio), IPO5 (sc-55,527, Santa Cruz), LaminA/C (10298-1-AP, Proteintech), Human-Reactive STING Pathway Antibody Sampler Kit (38,866, CST), FLAG (F1804, Sigma), HA (51064-2-AP, Proteintech), STAT1 (9172, CST), pSTAT1 (9167, CST), H3 (17168-1-AP, Proteintech), H3K9me3 (ab8898, Abcam), and SUV39H1 (05-615, Sigma).

Techniques: Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Over Expression, Immunofluorescence, Construct, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Nuclear translocation of ISG15 regulated by PPP2R2B inhibits cisplatin resistance of bladder cancer

doi: 10.1007/s00018-024-05320-1

Figure Lengend Snippet: Schematic diagram shows the mechanism that PPP2R2B mediates cisplatin sensitivity. PPP2R2B, which is down-regulated by SUV39H1-mediated trimethylation of H3K9, promotes entry of ISG15 into the nucleus by mediating the binding of IPO5 and ISG15. Nuclear translocation of ISG15 inhibited DNA repair, further increasing ISG15 expression through activation of the STING pathway

Article Snippet: Antibodies used for western blotting were as follows: PPP2R2B (ab157461, Abcam), GAPDH (60004-1-Ig, Proteintech), beta tubulin (10068-1-AP, Proteintech), phospho-DNA PKcs (Ser2056) (ab124918, Abcam), phospho-ATR (Ser428) (2853, CST), phospho-ATM (Ser1981) (5883, CST), ISG15 (15981-1-AP, Proteintech), anti-phosphoserine/threonine (PP2551, ECMbio), IPO5 (sc-55,527, Santa Cruz), LaminA/C (10298-1-AP, Proteintech), Human-Reactive STING Pathway Antibody Sampler Kit (38,866, CST), FLAG (F1804, Sigma), HA (51064-2-AP, Proteintech), STAT1 (9172, CST), pSTAT1 (9167, CST), H3 (17168-1-AP, Proteintech), H3K9me3 (ab8898, Abcam), and SUV39H1 (05-615, Sigma).

Techniques: Binding Assay, Translocation Assay, Expressing, Activation Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Differential Gene Expression Pattern of Importin β3 and NS5 in C6/36 Cells Acutely and Persistently Infected with Dengue Virus 2.

doi: 10.3390/pathogens12020191

Figure Lengend Snippet: Figure 3. Determination of viral genome copy number. It was determined by RT-qPCR in C6/36 infected by DENV-2 (C6/36 + D2) at an MOI of 0.1 (A), 2 (B), and 10 (C) for 12, 24, 36, 48, 72, and 120 h. The same experiment was performed in C6/36 cells persistently infected with DENV-2 (C6-L55) using equivalent amounts of brain extract from noninfected mice as inoculum. The experiments were performed in triplicate. The results were analyzed by two-way ANOVA and multiple comparison tests of Bonferroni. **** p < 0.0001.

Article Snippet: The protein extract was mixed with loading buffer (Tris-HCl, β-mercaptoethanol, SDS, bromophenol blue, glycerol) and boiled for 5 min. A total protein extract (30 μg when possible) was separated by 10% SDS–PAGE (Mini-Protean system, Bio-Rad, Hercules, CA, USA) as described previously [6] and electrotransferred onto a PVDF membrane (Immobilon®, Millipore, Burlington, MA, USA) using a Trans-Blot® semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations in Bjerrum-Schafer-Nielsen transfer buffer (Bjerrum and Schafer-Nielsen, 1986) at 0.8 mA/cm2 from 1 to 2 h. Immunoblotting was carried out using a mouse anti-karyopherin β3 (A-2) monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-514122), a mouse anti-β-actin (C4) monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-47778, RRID:AB_626632) was used as a loading control, and a rabbit anti-NS5 polyclonal antibody (Invitrogen, Waltham, MA, USA, PA5-32200, RRID AB_2549673) was diluted 1:80, 1:10,000 and 1:4000 respectively and incubated overnight at 4 ◦C.

Techniques: Quantitative RT-PCR, Infection, Comparison

Journal: Pathogens (Basel, Switzerland)

Article Title: Differential Gene Expression Pattern of Importin β3 and NS5 in C6/36 Cells Acutely and Persistently Infected with Dengue Virus 2.

doi: 10.3390/pathogens12020191

Figure Lengend Snippet: Figure 4. Expression of importin β3. It was determined by RT-qPCR in C6/36 infected by DENV- 2 (C6/36 + D2) at an MOI of 0.1 (A), 2 (B), and 10 (C) for 12, 24, 36, 48, 72, and 120 h. The same experiment was performed in C6/36 cells persistently infected with DENV-2 (C6-L55) using equivalent amounts of brain extract from noninfected mice as inoculum. All experiments were performed in triplicate. The results were analyzed by two-way ANOVA and multiple comparison tests of Bonferroni. **** p < 0.0001.

Article Snippet: The protein extract was mixed with loading buffer (Tris-HCl, β-mercaptoethanol, SDS, bromophenol blue, glycerol) and boiled for 5 min. A total protein extract (30 μg when possible) was separated by 10% SDS–PAGE (Mini-Protean system, Bio-Rad, Hercules, CA, USA) as described previously [6] and electrotransferred onto a PVDF membrane (Immobilon®, Millipore, Burlington, MA, USA) using a Trans-Blot® semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations in Bjerrum-Schafer-Nielsen transfer buffer (Bjerrum and Schafer-Nielsen, 1986) at 0.8 mA/cm2 from 1 to 2 h. Immunoblotting was carried out using a mouse anti-karyopherin β3 (A-2) monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-514122), a mouse anti-β-actin (C4) monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-47778, RRID:AB_626632) was used as a loading control, and a rabbit anti-NS5 polyclonal antibody (Invitrogen, Waltham, MA, USA, PA5-32200, RRID AB_2549673) was diluted 1:80, 1:10,000 and 1:4000 respectively and incubated overnight at 4 ◦C.

Techniques: Expressing, Quantitative RT-PCR, Infection, Comparison