Journal: Nature Communications

Article Title: Structural basis of arrestin-3 activation and signaling

doi: 10.1038/s41467-017-01218-8

Figure Lengend Snippet: IP mediated trimerization and receptor-independent activation in cells. a - b Sedimentation velocity analytical ultracentrifugation (SVAUC) of arrestin-3. Measurements were performed in triplicate. a Representative SVAUC run in the absence of IP predicts a molecular weight matching a monomer. b In the presence of 100 µM IP , the predicted molecular weight is consistent with a trimer. c Representative size exclusion chromatograms of arrestin-3 (Arr3) in the presence and absence of IP . In the absence of IP , 60 µM arrestin-3 (black) elutes at volume corresponding to the Stokes radius of a globular protein with a molecular mass of 64 ± 5.9 kDa. In the presence of 100 μM IP , the elution volume is consistent with a globular protein of molecular mass 169.6 ± 9.7 kDa. The ratio of these molecular weights is consistent with IP -dependent trimer formation. Measurements were performed in triplicate, errors are ± SEM. d Plot of the probability of the distances between spin labels at S13 and A392 for 100 µM arrestin-3 in the presence of the indicated molar ratios of IP ; inset shows the location of the spin labeled sites in basal arrestin-3 (PDB entry 3P2D ). e Comparison of JNK3 activation by GFP-arrestin-3 and Cys-less mutant. GFP-tagged arrestin allowed comparison of the expression levels of wild-type and variant arrestin-3, as described , . JNK3 activation (mean ± SEM) was assessed by measuring the pp-JNK3 levels in COS7 cells co-transfected with HA-ASK1, HA-JNK3 and GFP or Venus-tagged wild-type and Cys-less arrestin-3. Assay was repeated five times and JNK3 phosphorylation was compared by one-way ANOVA followed by Bonferroni post hoc test with correction for multiple comparisons. *** p < 0.001

Article Snippet: The expression level of HA-ASK1, Flag-JNK3 and arrestin-3 were assessed by Western analysis using antibodies against the HA tag (Cell Signaling #C29F4, 1:1000 dilution), the Flag tag (Sigma #F3165, 1:500 dilution), arrestin (F4C1) or GFP (JL-8, Choltech #632381, 1:2000 dilution), respectively.

Techniques: Activation Assay, Sedimentation, Analytical Ultracentrifugation, Molecular Weight, Labeling, Comparison, Mutagenesis, Expressing, Variant Assay, Transfection, Phospho-proteomics

Journal: Cancer Cell International

Article Title: Mcl-1 downregulation enhances BCG treatment efficacy in bladder cancer by promoting macrophage polarization

doi: 10.1186/s12935-025-03676-3

Figure Lengend Snippet: Mcl-1shRNA combined with BCG vaccine may promote the apoptosis of mouse bladder cancer MB49 cells by activating the ASK1/MKK7/JNK signaling pathway. (A) Apoptosis of MB49 cells in different groups of Hoechst (scale: 50 μm). (B) Hoechst statistical analysis of apoptosis of MB49 cells in different groups. (C , D) Flow cytometry was used to detect the apoptosis of MB49 cells in different groups and make statistics. (E) Expression of P-ASK1, ASK1, P-MKK7, MKK7, P-JNK, JNK, P-cJUN, cJUN and CX43 in MB49 cells. (F) P-ASK1, ASK1, P-MKK7, MKK7, P-JNK, JNK, P-cJUN, cJUN, and CX43 protein expression analysis. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with M0 + BCG-MB49 group; ## P < 0.01, ### P < 0.001 compared with M0 + BCG + Mcl-1shRNA-MB49 group

Article Snippet: Anti-JNK (1:5000, 66210-1-Ig), anti-ASK1 (1:3000, 67072-1-Ig), anti-CX43 (1:4000, 26980-1-AP) were purchased from Proteintech Biotechnology Co., LTD. (Wuhan, China).The following antibodies: Bax (1:1000, bs-4605R), Bcl-2 (1:1000, bsm-52304R), P-ASK1(1:3000, bs-3029R), MKK7 (1:1000, bs-1979R), P-MKK7 (1:300, bs-3277R), P-JNK (1:500, bs-1640R), cJUN (1:1000, bs-0670R), P-cJUN (1:1000, bsm-52141R), CyclinD1 (1:1000, bsm-52046R) and PCNA (1:1000, bsm-52347R), were obtained from Bioss Biotechnology Co., LTD. (Beijing, China).

Techniques: Flow Cytometry, Expressing

Journal: Cancer Cell International

Article Title: Mcl-1 downregulation enhances BCG treatment efficacy in bladder cancer by promoting macrophage polarization

doi: 10.1186/s12935-025-03676-3

Figure Lengend Snippet: Mcl-1shRNA combined with BCG vaccine may treat bladder cancer in rats by activating the ASK1/MKK7/JNK signaling pathway. (A) Immunofluorescence detection of ASK1 and P-ASK1 expression in bladder tissue of rats in each group (scale: 50 μm). (B) Immunofluorescence detection of MKK7 and P-MKK7 expression in bladder cancer tissues of rats in each group (scale: 50 μm). (C) Immunofluorescence detection of the expression of JNK and P-JNK in bladder cancer tissues of rats in each group (scale: 50 μm). (D) Immunofluorescence detection of cJUN and P-CJUN expression in bladder cancer tissues of rats in each group (scale: 50 μm)

Article Snippet: Anti-JNK (1:5000, 66210-1-Ig), anti-ASK1 (1:3000, 67072-1-Ig), anti-CX43 (1:4000, 26980-1-AP) were purchased from Proteintech Biotechnology Co., LTD. (Wuhan, China).The following antibodies: Bax (1:1000, bs-4605R), Bcl-2 (1:1000, bsm-52304R), P-ASK1(1:3000, bs-3029R), MKK7 (1:1000, bs-1979R), P-MKK7 (1:300, bs-3277R), P-JNK (1:500, bs-1640R), cJUN (1:1000, bs-0670R), P-cJUN (1:1000, bsm-52141R), CyclinD1 (1:1000, bsm-52046R) and PCNA (1:1000, bsm-52347R), were obtained from Bioss Biotechnology Co., LTD. (Beijing, China).

Techniques: Immunofluorescence, Expressing

Journal: Cancer Cell International

Article Title: Mcl-1 downregulation enhances BCG treatment efficacy in bladder cancer by promoting macrophage polarization

doi: 10.1186/s12935-025-03676-3

Figure Lengend Snippet: Mcl-1 shRNA combined with BCG vaccine may promote the apoptosis of bladder cancer cells by activating the ASK1/MKK7/JNK signaling pathway and enhance the efficacy of treating bladder cancer in rats. (A) Tunel staining of rat bladder tissue in each group (scale: 100 μm). (B) Protein band diagrams of Bax and Bcl-2 in bladder cancer tissues of rats in each group. (C , D) Protein expression analysis of CyclinD1 and PCNA in bladder cancer tissues of rats in each group. (E) Immunohistochemistry detects the expression of P-JNK, P-cJUN, and CX43 in the bladder tissue of rats in each group (scale: 50 μm). (F) cJUN, P-CJUN and CX43 protein band diagrams in bladder cancer tissues of rats in each group. (G) Protein band diagrams of P-JNK and JNK in bladder cancer tissues of rats in each group. (H) Analysis of cJUN and P-CJUN protein expression in bladder cancer tissues of rats in each group. (I) Analysis of JNK and P-JNK protein expression in bladder cancer tissues of rats in each group. (J) CX43 protein expression analysis in bladder cancer tissues of rats in each group. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with BLCA group; & P < 0.05, && P < 0.01, &&& P < 0.001 compared with BCG group; $ P < 0.05, $$ P < 0.01 compared with BCG + Mcl-1shRNA group

Article Snippet: Anti-JNK (1:5000, 66210-1-Ig), anti-ASK1 (1:3000, 67072-1-Ig), anti-CX43 (1:4000, 26980-1-AP) were purchased from Proteintech Biotechnology Co., LTD. (Wuhan, China).The following antibodies: Bax (1:1000, bs-4605R), Bcl-2 (1:1000, bsm-52304R), P-ASK1(1:3000, bs-3029R), MKK7 (1:1000, bs-1979R), P-MKK7 (1:300, bs-3277R), P-JNK (1:500, bs-1640R), cJUN (1:1000, bs-0670R), P-cJUN (1:1000, bsm-52141R), CyclinD1 (1:1000, bsm-52046R) and PCNA (1:1000, bsm-52347R), were obtained from Bioss Biotechnology Co., LTD. (Beijing, China).

Techniques: shRNA, TUNEL Assay, Staining, Expressing, Immunohistochemistry