Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: Transcriptome sequencing analysis and pathway enrichment analysis. Volcano plot illustrating the results of differential gene analysis between the PMEPA1 knockdown group and the control group of MKN-7 cells in transcriptome sequencing ( A ). GO enrichment analysis of differentially expressed genes reveals prominent enrichment in the biological process of cell cycle ( B ). Gene Set Enrichment Analysis (GSEA) indicated predominant upregulation of gene expression in the cell cycle pathway, with prominent representation among the top-ranked differentially expressed genes ( C ). The intersection of proteins detected exclusively in the overexpression group through mass spectrometry analysis and the set of differentially expressed genes yielded the identification of SFN (14-3-3σ) ( D ).

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: Sequencing, Knockdown, Control, Gene Expression, Over Expression, Mass Spectrometry

Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: Expression patterns and prognostic values of PMEPA1 both in public databases (TCGA and GEO) and Xiangya cohort. PMEPA1 expression was analyzed in unpaired ( A ) and paired ( B ) TCGA-STAD cohorts, and the GSE26899 cohort ( C ). Kaplan-Meier plots for overall survival (OS) and disease-free survival in the TCGA-STAD cohort ( D , E ), OS in the GSE84437 ( F ), and Xiangya Hospital cohorts ( I ) were grouped by PMEPA1 expression. PMEPA1 expression comparison between cancer and adjacent normal tissues ( G, scale bar 200 μm) and its relationship with pathological staging and scoring in the Xiangya cohort (166 patients) ( H, data are reported as median and interquartile range) were also examined. Wilcoxon rank-sum test was used for P-values in A and C , paired Wilcoxon test in B , and log-rank test in I (*P<0.05, ****P<0.0001). Long-term survival differences in D and E , and OS differences in F , were assessed using partial and regular log-rank tests, respectively.

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: Expressing, Comparison

Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: Statistical analysis of the correlation between PMEPA1 and clinicopathological data.

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: Expressing

Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: In vitro cellular functional experiments of PMEPA1 overexpression and knockdown. Stable cell lines overexpressing PMEPA1 in AGS (OE-PMEPA1) and control (vector), as well as PMEPA1 knockdown (sh-PMEPA1) and control (scramble) in MKN-7, were established and then validated by western blot ( A ). MTT assay was applied to detect cell viability of stably transfected AGS and MKN-7 cells ( B ). Clone formation assay was used to determine cell proliferation of stably transfected AGS and MKN-7 cells ( C ). Transwell assay and wound healing assay were applied to detect cell migration of stably transfected AGS and MKN-7 cells ( D and E ). Flow cytometry was used to ascertain cell cycle of stably transfected MKN-7 cells ( F ). Data are reported as means±SD. **P<0.01, ***P<0.001 compared to vector or scramble group; Student's t -test.

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: In Vitro, Functional Assay, Over Expression, Knockdown, Stable Transfection, Control, Plasmid Preparation, Western Blot, MTT Assay, Transfection, Tube Formation Assay, Transwell Assay, Wound Healing Assay, Migration, Flow Cytometry

Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: Validation of the oncogenic function of PMEPA1 in vivo . PMEPA1-overexpressing AGS cells, PMEPA1-silenced MKN-7 cells, and their respective control cells were subcutaneously injected into NOD-SCID mice and then the mice were sacrificed, and tumors were measured and are shown at post-30-day ( A and E ). Tumor weights were recorded post-excision ( B and F ). Tumor volumes were measured every 5 days, generating growth curves ( C and G ). Tumor tissues were analyzed for PMEPA1 expression via western blot ( D and H ), and a portion was immunohistochemically examined for PMEPA1 and Ki67 expression ( I , scale bar 200 μm). Data are reported as means±SD. *P<0.05, **P<0.01 compared to vector or scramble group; Student's t -test.

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: Biomarker Discovery, In Vivo, Control, Injection, Expressing, Western Blot, Plasmid Preparation

Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: PMEPA1 influenced cell proliferation and cell cycle through the 14-3-3σ/cdc25c signaling axis. After knocking down PMEPA1 in MKN-7 cells, changes in the expression and phosphorylation levels of 14-3-3σ, downstream Cdc25c, and the cdk1/cyclin B complex were observed by western blot ( A ). The shRNA vector of PMEPA1 and 14-3-3σ were transfected into MKN-7 cells, and the expression levels of PMEPA1 and 14-3-3σ proteins were detected by western blot ( B ). The cell cycle distribution ( C ), cell proliferation ability ( D ), and colony formation capacity ( E ) were assessed after the double knockdown of PMEPA1 and 14-3-3σ using flow cytometry, MTT, and clone formation assays, respectively. Data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001, ns: non-significant compared to scramble group; Student's t -test.

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: Expressing, Phospho-proteomics, Western Blot, shRNA, Plasmid Preparation, Transfection, Knockdown, Flow Cytometry

Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: PMEPA1 interacted with 14-3-3σ and affected its stability. In AGS cells (exogenous) and MKN-7 cells (endogenous), the interaction between PMEPA1 and 14-3-3σ was validated using co-immunoprecipitation (co-IP) ( A ). The interaction between PMEPA1 and 14-3-3σ in AGS and MKN-7 cells was further confirmed through immunofluorescence assays ( B , scale bar 20 μm). The stability of 14-3-3σ expression following overexpression or knockdown of PMEPA1 in AGS or MKN-7 cells was assessed by western blot after treating the cells with cycloheximide (CHX, 20 μg/mL) for 0, 4, 8, and 12 h ( C ). The changes in mRNA levels of 14-3-3σ after overexpression or knockdown of PMEPA1 in AGS or MKN-7 cells were detected by quantitative PCR (qPCR) ( D ). The impact of overexpression or knockdown of PMEPA1 on the ubiquitination levels of 14-3-3σ was examined through co-IP ( E ). After treating the PMEPA1 overexpression AGS cells or PMEPA1 knockdown MKN-7 cells with MG132 (25 μM) for 24 h, the reversal of 14-3-3σ degradation was evaluated by western blot ( F ). Data are reported as means±SD. **P<0.01, ***P<0.001, ns: non-significant; Student's t -test.

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Expressing, Over Expression, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Brazilian Journal of Medical and Biological Research

Article Title: PMEPA1 promotes gastric cancer cell proliferation by regulating the ubiquitin-mediated degradation of 14-3-3σ and promoting cell cycle progression

doi: 10.1590/1414-431X2024e13985

Figure Lengend Snippet: PMEPA1 recruited TTC3 to facilitate the ubiquitination of 14-3-3σ. Co-IP assays demonstrated the impact of PMEPA1 expression changes on the binding of TTC3 to 14-3-3σ in AGS and MKN-7 cells ( A ). Immunoblotting revealed that TTC3 siRNA reversed the degradation level of 14-3-3σ following the overexpression of PMEPA1. Moreover, even with the overexpression of TTC3, the knockdown of PMEPA1 also reversed the degradation level of 14-3-3σ ( B ). Immunofluorescence staining indicated that 14-3-3σ can bind with TTC3 ( C , scale bar 20 μm). Co-IP assays showed that siTTC3 reversed the ubiquitination of 14-3-3σ following the overexpression of PMEPA1, and similarly, even with the overexpression of TTC3, the knockdown of PMEPA1 also reversed the ubiquitination of 14-3-3σ ( D ).

Article Snippet: The sections were blocked and incubated with primary antibodies against PMEPA1 (1:200, 16521-1-AP, Proteintech, China) and Ki67 (1:2000, 27309-1-AP, Proteintech) overnight at 4°C.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Expressing, Binding Assay, Western Blot, Over Expression, Knockdown, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: STAG2 regulates interferon signaling in melanoma via enhancer loop reprogramming

doi: 10.1038/s41467-022-29541-9

Figure Lengend Snippet: a Circos plot for differential peaks of CTCF, SMC1A, STAG1, and STAG2 binding upon STAG2 knockdown in M14 cells. Red bar represents gain of binding and blue bar represents loss of binding. b Distinct profiles of changes in STAG1 binding at the loss of STAG2 binding sites upon STAG2 knockdown in M14 cells. The STAG switch and non-switch groups are identified by k-means clustering and show significant gain or nearly no gain in STAG1 binding at the loss of STAG2 binding sites, respectively. c Heatmap of normalized ChIP-seq signals for STAG1, STAG2, SMC1A, and CTCF in M14 cells with (KD) and without (WT) STAG2 shRNA knockdown and their changes (Δ), as defined by log 2 (KD/WT), in both STAG switch (cluster 1) and non-switch groups (cluster 2). d Average contact probability at a different genomic distance for KD and WT, using 100 kb as window size. e Average TAD length is significantly different between STAG2 WT ( n = 3789 TADs) and KD ( n = 3496 TADs). The box plot is defined by bounds at the 25th percentile and 75th percentile, center at 50th percentile, the minima and maxima are at the 10th percentile and 90th percentile. P value is 3.488e-13 and is based on a two-sided Wilcox test. f Difference in TAD size for expanded TADs ( n = 1125 TADs) is significantly longer than those of shrinked TADs ( n = 1007 TADs). P value is 2.312e-17 and is based on a two-sided Wilcox test. g CTCF, SMC1A, STAG1, and STAG2 profiles at stable and variable TAD boundaries in M14 STAG2 WT cells. RPM reads per million. h Remarkable STAG2 to STAG1 switch occurs at boundaries of expanded TADs but not shrinked TAD upon STAG2 knockdown. The schematic diagram depicts that the variable boundary (VB) start site is defined as the site in WT, and the related end site as the site in KD. Statistical significance is determined as: ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies against STAG2 (Santa Cruz, SC-81852), IRF9 (Cell Signaling, #76684), IRF7 (Cell Signaling, #4920), USP18 (Cell Signaling, #4813), ISG15 (Santa Cruz, SC-166755), IRF1 (Cell Signaling, #8478), IRF3 (Cell Signaling, #11904), GAPDH (Cell Signaling, #2118), GAPDH (Cell Signaling, #51332), STAG1 (Novus Biologicals, NB100-298), PD-L1 (R&D Systems, AF156), CTCF (Cell Signaling, #2899), and Flag M2 (Sigma, F3165) were used.

Techniques: Binding Assay, Knockdown, ChIP-sequencing, shRNA

Journal: Nature Communications

Article Title: STAG2 regulates interferon signaling in melanoma via enhancer loop reprogramming

doi: 10.1038/s41467-022-29541-9

Figure Lengend Snippet: a Heatmap representation of differential H3K27Ac-associated loops between M14 cells with (KD) and without (WT) STAG2 shRNA knockdown as indicated by black arrows. b Majority of loop changes upon STAG2 knockdown occurs within TADs. c Motifs of transcription factors enriched at anchors of enhanced and impaired loops. d Numbers of gained, lost, and maintained super-enhancers upon STAG2 knockdown in M14 cells. e Gain of H3K27ac signal at the STAG2 to STAG1 switch sites, but not non-switch sites, upon STAG2 knockdown. f Differential CTCF, SMC1A, STAG1, STAG2, and H3K27ac profiles at anchors of enhanced and impaired loops upon STAG2 knockdown. g The relative gain of STAG1 in compensation to the loss of STAG2 is significantly higher at anchors of enhanced loops than those of impaired loops. The degree of the switch is calculated as the sum of log 2 (KD/WT) for STAG1 and STAG2 (method).

Article Snippet: The primary antibodies against STAG2 (Santa Cruz, SC-81852), IRF9 (Cell Signaling, #76684), IRF7 (Cell Signaling, #4920), USP18 (Cell Signaling, #4813), ISG15 (Santa Cruz, SC-166755), IRF1 (Cell Signaling, #8478), IRF3 (Cell Signaling, #11904), GAPDH (Cell Signaling, #2118), GAPDH (Cell Signaling, #51332), STAG1 (Novus Biologicals, NB100-298), PD-L1 (R&D Systems, AF156), CTCF (Cell Signaling, #2899), and Flag M2 (Sigma, F3165) were used.

Techniques: shRNA, Knockdown

Journal: Oncogene

Article Title: CRISPR-based dissection of microRNA-23a ~ 27a ~ 24-2 cluster functionality in hepatocellular carcinoma

doi: 10.1038/s41388-024-03115-z

Figure Lengend Snippet: A – C Interaction analysis of miR-23a-5p or miR-27a-5p mimic with the 3′-UTRs of PURA, STAG1 , and TTK mRNA by miRNA/mRNA IP assay of HepG2 cells. Data are presented as means ± SD. *** p < 0.001 by two-tailed t -test vs. Scr group. D mRNA expression of PURA measured by qPCR in Scr, miR-23a KO, or miR-27a KO HepG2 cells treated with or without miRNA mimic or inhibitor. E Protein expression of PURA measured by Western blot in Scr, miR-23a KO, or miR-27a KO HepG2 cells. F Protein expression of PURA by IHC staining in xenograft tumors injected with Scr, miR-23a KO, and miR-27a KO HepG2 cells in NSG mice. Scale bars=50μm. G , H Luciferase reporter activity in HepG2 cells co-transfected with Scr vector, wild-type or mutant 3’-UTR PURA constructs with mimics or inhibitor for miR-23a-3p or miR-27a-3p. *** p < 0.001 by one-way ANOVA Tukey’s multiple comparisons test vs . Scr group. I Protein expression of PURA measured by Western blot in Scr cells or cells transfection with PURA siRNA. J , K Cell growth curve in Scr, miR-23a KO, miR-23a KO, miR-27a KO, and miR-27a KO cells transfected with PURA siRNA for 6 days. Data are presented as means ± SD. * p < 0.05 and *** p < 0.001 by two-way ANOVA test vs . Scr or PURA siRNA group. L Protein expression of STAG1 and TTK measured by Western blot in Scr, miR-23a KO or miR-27a KO transfected with PURA siRNA after 48 h, respectively. M Protein expression of STAG1 and TTK measured by Western blot in Scr, miR-23a KO, miR-23a KO + PURA siRNA, Scr+miR-23a-3p mimic, Scr+miR-23a-3p mimic+ PURA siRNA, miR-27a KO, miR-27a KO + PURA siRNA, Scr+miR-27a-3p mimic, and Scr+miR-27a-3p mimic+ PURA siRNA cells, respectively. All experiments were repeated three times.

Article Snippet: Antibodies specific for the following proteins were used as primary antibodies for Western blot or IHC: Beta catenin (ab32572, 1:5000, Abcam, Cambridge, MA), beta IV Tubulin (ab179509, 1:5000, Abcam), CDC27 (A1954, 1:1000, Abclonal, Woburn, MA), BMPR2 (A16778, 1:1000, Abclonal), CDK1 (626901, 1:1000, Biolegend, San Diego, CA), CDK2 (643901, 1:1000, Biolegend), CDK4 (2906, 1:1000, Cell Signaling, Danvers, MA), c-Myc (5605, 1:1000, Cell Signaling), Cyclin A (644001, 1:1000, Biolegend), PURA (A9296, 1:1000 for Western blot and 1:100 for IHC, Abclonal), Cyclin B (647902, 1:1000 for Western blot and 1:100 for IHC, Biolegend), Cyclin D1 (ab134175, 1:5000, Abcam), Cyclin E (sc-247, 1:1000, Santa Cruz Biotechnology, Dallas, TX), E-cadherin (3195, 1:1000 for Western blot and 1:100 for IHC, Cell Signaling), GAPDH (2118, 1:5000, Cell Signaling), Ki67 (ab15580, 1:5000 for Western blot and 1:200 for IHC, Abcam), Lamin B (sc-374015, 1:5000, Santa Cruz Biotechnology), N-cadherin (844702, 1:1000 for Western blot and 1:100 for IHC, Biolegend), p21 (2947, 1:1000, Cell Signaling), p53 (sc-126, 1:1000, Santa Cruz Biotechnology), phospho-cdc2 (Tyr15) (4539, 1:1000 for Western blot and 1:100 for IHC, Cell Signaling), phospho-Smad1/Smad5/Smad9 (13820, 1:1000, Cell Signaling), Smad1 (6944, 1:1000, Cell Signaling), Snail (ab167609, 1:5000, Abcam), STAG1 (A13715, 1:1000, Abclonal), TMEM170B (NBP2-33739, 1:1000 NOVUS Biologicals, Centennial CO), TTK (A2500, 1:1000, Abclonal), Twist (ab175430, 1:5000, Abcam), Vimentin (sc-6260, 1:1000, Santa Cruz Biotechnology), and ZEB1 (3396, 1:1000, Cell Signaling).

Techniques: Two Tailed Test, Expressing, Western Blot, Immunohistochemistry, Injection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Construct

Journal: eLife

Article Title: Precision RNAi using synthetic shRNAmir target sites

doi: 10.7554/eLife.84792

Figure Lengend Snippet: ( A ) Schematic of EGFR del19 ::V5::dsRed::ARTi engineering in PC-9 cells. Blue color denotes overexpressed ARTi variant. Green denotes endogenous gene. ( B ) Western blot demonstrating knockdown of EGFR del19 ::V5::dsRed::ARTi. Western blot is a representative example of three independent biological repeat experiments. ( C ) Proliferation assay and crystal violet staining of parental and engineered PC-9 cells in the absence or presence of doxycycline (dox). Crystal violet staining is a representative example of two independent biological repeat experiments.( D ) In vivo experiment comparing dox-induced EGFR del19 ::V5::dsRed::ARTi knockdown to pharmacological EGFR del19 inhibition. Mean tumor volume and ± SEM is plotted for all in vivo experiments. ( E ) Schematic of MIA PaCa-2 engineering. Blue color denotes overexpressed ARTi variant. Green denotes endogenous gene. ( F ) Western blot for KRAS and Actin in indicated engineered MIA PaCa-2 cells in the presence and absence of dox. Western blot is a representative example of two independent biological repeat experiments. ( G ) Proliferation assay and crystal violet staining of parental and engineered MIA PaCa-2 cells in the absence or presence of dox. Crystal violet staining is a representative example of two independent biological repeat experiments. ( H ) Growth curves of tumors implanted with engineered MIA PaCa-2 cells in the absence and presence of dox in vivo. ( I ) Schematic of C-terminal endogenous tagging of STAG1. Green color denotes endogenous genes. ( J ) Western blot demonstrating knockdown of STAG1-ARTi. Western blot is a representative example of three independent biological repeat experiments. ( K ) Immunohistochemistry staining of STAG1 in engineered HCT 116 cells in the absence and presence of dox. Asterisk marks an area of murine fibroblasts that serve as an internal positive control. ( I ) Growth curves of tumors implanted with engineered HCT 116 cells in the absence and presence of dox. Figure 2—source data 1. Original blots for and . Figure 2—source data 2. Original blots for . Figure 2—source data 3. Original blots for .

Article Snippet: The following primary antibodies were used for immunoblot analyses: EGFR (Cell Signaling, #4267); STAG1 (GeneTex, GTX129912); STAG2 (Bethyl, A300-159A); b-actin (Sigma, A5441); KRAS (LSbio, LS-C17566); and V5 (Sigma, V8012).

Techniques: Variant Assay, Western Blot, Knockdown, Proliferation Assay, Staining, In Vivo, Inhibition, Immunohistochemistry, Positive Control

Journal: eLife

Article Title: Precision RNAi using synthetic shRNAmir target sites

doi: 10.7554/eLife.84792

Figure Lengend Snippet: ( A ) Schematic of synthetic lethal interaction between STAG1 and STAG2. Cells survive loss of either paralog but are incapable of growing upon combined loss of STAG1 and STAG2. ( B ) Western blot confirmation of STAG2 knockout and ARTi-shRNAmir-induced knockdown of endogenous STAG1::V5::ARTi. Western blot is a representative example of three independent biological repeat experiments. ( C ) Proliferation assay of ARTi engineered HCT 116 cells in vitro, visualized by crystal violet staining following a 9-day doxycycline (dox) or control treatment. Staining is a representative example of three independent biological repeat experiments. ( D ) Quantification of nuclear Stag1 level using engineered HCT 116 cells in an in vivo xenotransplantation experiment the control group (-dox) and dox (+dox) treated group. Figure 2—figure supplement 3—source data 1. Original blots for .

Article Snippet: The following primary antibodies were used for immunoblot analyses: EGFR (Cell Signaling, #4267); STAG1 (GeneTex, GTX129912); STAG2 (Bethyl, A300-159A); b-actin (Sigma, A5441); KRAS (LSbio, LS-C17566); and V5 (Sigma, V8012).

Techniques: Western Blot, Knock-Out, Knockdown, Proliferation Assay, In Vitro, Staining, Control, In Vivo

Journal: Journal of Virology

Article Title: Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements

doi: 10.1128/JVI.02231-17

Figure Lengend Snippet: siRNA knockdown of SV40-sT/LTag expression in 293T cells. (A) Experimental timeline of transfection and flow-cytometric analysis (fluorescence-activated cell sorter [FACS]). (B) Immunoblot demonstrating efficient knockdown of LTag protein and sTag protein in 293T cells transfected with siRNA (sT/LTAG or scrambled) for up to 72 h. Immunoblotting against the actin protein was used as a loading control. (C) Mean fluorescence intensity of the indicated NCCR reporter constructs.

Article Snippet: The membrane was dried, reactivated with 5 ml methanol (Sigma-Aldrich, Switzerland), and washed twice with milli-Q H 2 O. Odyssey blocking buffer (927-40000; Licor, Lincoln, NE, USA) diluted 1:2 in Tris-buffered saline (TBS) was used to block the membrane at RT for 1 h. Incubation of the membrane was done with the following primary antibodies: monoclonal mouse anti-actin (1:5,000; Abcam, Cambridge, England), mouse IgG2 anti-LTag cross-reacting with sTag (1:500; BD Pharmingen), diluted in 1:2 Odyssey blocking buffer–TBS–0.1% Tween 20 at RT for 2 h. The membrane was then washed at least 5 times with TBS–0.1% Tween 20.

Techniques: Expressing, Transfection, Fluorescence, Western Blot, Construct