53484s primary antibodies  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: RECODE: a programmable guide-free C-to-U RNA editing tool

doi: 10.1093/nar/gkaf1309

Figure Lengend Snippet: Influence of the PPR domain length on RECODE-PG and RECODE-WW protein activity. ( A ) Sequences of nine selected off-target editing sites aligned with the CTNNB1-T41I on-target site. The arrow indicates the editing site. ( B–G ) Analysis of the influence of the PPR domain length (10, 12, 14, and 16 P-motifs) on RECODE-PG [panels (B), (C), and (F)] and RECODE-WW [panels (D), (E), and (G)] activity in HEK293T cells. ( B, D ) On-target editing activity at the endogenous CTNNB1-T41I site. ( C, E ) Off-target editing activity. ( F, G ) β-catenin protein accumulation levels, normalized to total protein and subsequently to the empty vector samples. Data are presented as the mean ± standard deviation (SD) of three biological replicates ( n = 3). Each dot corresponds to one replicate. Letters indicate significant differences (one-way ANOVA, Tukey’s comparison test, P <.05) between the different lengths of the PPR domain. Multiple letters indicate no significant differences.

Article Snippet: Primary antibodies against β-catenin (sc-59737, Santa Cruz Biotechnology; 1:250), KRAS (12063-1-AP, Proteintech; 1:200), and BRG1/SMARCA4 (21634-1-AP, Proteintech; 1:100) were used.

Techniques: Activity Assay, Plasmid Preparation, Standard Deviation, Comparison

Journal: Nucleic Acids Research

Article Title: RECODE: a programmable guide-free C-to-U RNA editing tool

doi: 10.1093/nar/gkaf1309

Figure Lengend Snippet: Optimization of RECODE design in HEK293T cells. ( A ) Amino acid sequence alignment of the E domains of RECODE-PG and RECODE-WW. Sequences are compared to a consensus sequence derived from the alignment of PG and WW E domains from hornwort, lycophyte and fern transcriptomes. The fifth (5) and last (L) amino acid positions in the E1 motif that are involved in specific nucleotide recognition are shown. Grey underlines indicate the first helix region of each E motif. Amino acids identical to the consensus sequence are indicated by a dot. ( B–E ) Evaluation of the influence of amino acid mutations in the E domain on the activity in HEK293T cells of RECODE-PG ( B, C ) and RECODE-WW ( D, E ) containing 14 P-motifs. ( B, D ) Editing efficiency on the endogenous CTNNB1-T41I site. ( C, E ) β-catenin protein accumulation driven by RNA editing in RECODE mutants, normalized to total protein and subsequently to empty vector samples. ( F–K ) Analysis of the influence of varying PPR-domain lengths (10, 12, 14, and 16 P-motifs) on RECODE-PG 2 [panels (F), (G), and (J)] and RECODE-WW 2 [panels (H), (I), and (K)] in HEK293T cells. ( F, H ) On-target editing activity at the CTNNB1-T41I site. ( G, I ) β-catenin protein accumulation levels, normalized to total protein and subsequently to empty vector samples. Data are presented as the mean ± SD of three biological replicates ( n = 3). ( J, K ) Off-target editing activity. Each dot corresponds to one replicate. Letters indicate significant differences (one-way ANOVA, Tukey’s comparison test, P <.05) between the different lengths of the PPR domain. Multiple letters indicate no significant differences.

Article Snippet: Primary antibodies against β-catenin (sc-59737, Santa Cruz Biotechnology; 1:250), KRAS (12063-1-AP, Proteintech; 1:200), and BRG1/SMARCA4 (21634-1-AP, Proteintech; 1:100) were used.

Techniques: Sequencing, Derivative Assay, Activity Assay, Plasmid Preparation, Comparison

Journal: Nucleic Acids Research

Article Title: RECODE: a programmable guide-free C-to-U RNA editing tool

doi: 10.1093/nar/gkaf1309

Figure Lengend Snippet: Time-course analysis of RECODE variants. ( A ) Cytotoxicity, relative to cells transfected with an empty vector, was evaluated using an LDH assay at 24, 48, and 72 h. Each data point represents the mean value ± SD of three biological replicates ( n = 3). ( B ) Editing efficiency of CTNNB1-T41I site over time. Asterisks denote significant differences between each time point (* P <.05 and ** P <.01; one-way ANOVA, Tukey’s comparison test). ( C ) Relative β-catenin protein accumulation levels, normalized to total protein and subsequently to empty vector samples. Letters indicate significant differences between protein variants (one-way ANOVA, Tukey’s comparison test, P <.05). Multiple letters indicate no significant difference. ( D ) Relative CTNNB1 mRNA expression levels, normalized to GAPDH gene and subsequently to empty vector samples. Asterisks denote significant differences compared to the empty vector control (* P <.05; Welch’s t -test). ( B–D ) All data are presented as the mean ± SD of three biological replicates ( n = 3). Each data point corresponds to a single biological replicate.

Article Snippet: Primary antibodies against β-catenin (sc-59737, Santa Cruz Biotechnology; 1:250), KRAS (12063-1-AP, Proteintech; 1:200), and BRG1/SMARCA4 (21634-1-AP, Proteintech; 1:100) were used.

Techniques: Transfection, Plasmid Preparation, Lactate Dehydrogenase Assay, Comparison, Expressing, Control

Journal: Journal of Ovarian Research

Article Title: Hyptolide induces ER stress-mediated cell death and enhances GSK3β-regulated cisplatin chemosensitivity in ovarian cancer

doi: 10.1186/s13048-025-01712-4

Figure Lengend Snippet: Hyptolide inhibits nuclear translocation of β-catenin in cisplatin-resistant ovarian cancer cells. WT, CP1#1, and CP1#2 cells were treated with 10 μM hyptolide for 0, 2, 4, and 12 h. A Immunofluorescence staining of β-catenin (green) and DAPI nuclear stain (blue) was performed and fluorescence images were captured using confocal microscopy (FV3000, OLYMPUS). B Quantification of the nuclear and cytoplasmic fluorescence intensities of β-catenin. The nuclear–cytoplasmic ratio (N/C ratio) of β-catenin was quantified and calculated using ImageJ software. ( C ) Representative blots and ( D ) quantification of phosphorylated β-catenin (p-β-catenin Tyr654 ), β-catenin, and GAPDH. Analyses were conducted using three independent replicates with the bars representing SEM. Data were found to be significant at * p < 0.05, *** p < 0.001

Article Snippet: Then, cells were blocked using Invitrogen's CAS Blocking Histochemical Reagent for 1 h at room temperature, followed by overnight incubation with β-catenin primary antibodies (sc-7199, Santa Cruz).

Techniques: Translocation Assay, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy, Software

Journal: Journal of Ovarian Research

Article Title: Hyptolide induces ER stress-mediated cell death and enhances GSK3β-regulated cisplatin chemosensitivity in ovarian cancer

doi: 10.1186/s13048-025-01712-4

Figure Lengend Snippet: Schematic of the molecular mechanism of hyptolide acting on ovarian cancer cells. A Hyptolide induces cell apoptosis in WT and cisplatin-resistant ovarian cancer cells through induction of ER stress. B Hyptolide inhibits the activity of β-catenin and increases the expression of E-cadherin by activating GSK3β in cisplatin-resistant ovarian cancer cells

Article Snippet: Then, cells were blocked using Invitrogen's CAS Blocking Histochemical Reagent for 1 h at room temperature, followed by overnight incubation with β-catenin primary antibodies (sc-7199, Santa Cruz).

Techniques: Activity Assay, Expressing