Journal: Viruses

Article Title: The C4 Protein of TbLCYnV Promotes SnRK1 β2 Degradation Via the Autophagy Pathway to Enhance Viral Infection in N. benthamiana

doi: 10.3390/v16020234

Figure Lengend Snippet: TbLCYnV C4 promotes the degradation of NbSnRK1 β2 via the autophagy pathway. ( A ) Western blot analysis and semi-qRT-PCR assay of NbSnRK1 β2 protein and mRNA levels. N. bent hamiana leaves co-expressed NbSnRK1 β2-GFP with C4-mCherry or mCherry, respectively. Samples were analyzed by immunoblot using anti-GFP or anti-mCherry antibody. The amount of NbSnRK1 β2-GFP expression was calculated by ImageJ 1.8.0, and the data of NbSnRK1 β2-GFP co-infiltrated with mCherry were set as 1.00. Ponceau Red Stained Rubisco was used as a loading control. Semi-qRT-PCR assay was used to detect the transcription levels of NbSnRK1 β2 in plants. The expression of Actin gene was used as an internal control. ( B ) The expression of NbSnRK1 β2-GFP was calculated by GraphPad Prism 8. The data are shown as means and SD of three biological replicates. ** indicates a significant difference between the two treatments at the p < 0.01 by Student’s t -test. ( C ) Protease inhibitor treatment of NbSnRK1 β2 by Western blot. The N. benthamiana leaves transiently expressing NbSnRK1 β2-GFP were treated with 100 μM CHX together with 100 μM E64d, 5 mM 3-MA, 100 μM MG132, or DMSO, respectively. Samples were analyzed by immunoblot using anti-GFP antibody. The relative accumulation level of NbSnRK1 β2 in DMSO treatments were set as 1.00. Ponceau Red Stained Rubisco was used as a loading control. ( D ) The expression levels of NbSnRK1 β2-GFP by Western blot. The N. benthamiana leaves transiently co-expressing C4-mCherry or mCherry with NbSnRK1 β2-GFP were treated with 100 μM E64d or DMSO, respectively. The amount of NbSnRK1 β2-GFP expression was calculated using ImageJ 1.8.0. The relative accumulation level of NbSnRK1 β2 in the co-infiltrated with NbSnRK1 β2 and mCherry plants with DMSO treatment was set at 1.00. Ponceau Red Stained Rubisco was used as a loading control. ( E ) Semi-in-vivo protein stability assay of NbSnRK1 β2 by Western blot. NbSnRK1 β2 was transiently co-expressed with C4 (D22A)-mCherry or C4-mCherry in N. benthamiana plants, and E64d treatment was performed 48 h later. The data of NbSnRK1 β2-GFP co-infiltrated with C4-mCherry were set as 1.00. Ponceau Red Stained Rubisco was used as a loading control.

Article Snippet: MG132 (a specific 26S proteasome inhibitor) (MCE, Shanghai, China), 3-MA (an inhibitor of autophagy via its inhibitory effect on class III PI3K) (MCE), and E64d (a cysteine protease inhibitor associated with autophagy) (MCE) (all dissolved in DMSO) were diluted in ddH 2 O to 100 μM, 5 mM, and 100 μM, respectively [ , , ].

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Staining, Control, Protease Inhibitor, In Vivo, Stability Assay

Journal: Advanced Science

Article Title: F‐Box and Leucine‐Rich Repeat Protein 4 (FBXL4) Maintains Sarcomere Integrity and Cardiac Function by Enhancing K48‐Linked Ubiquitinated Degradation of Profilin‐1 (PFN1)

doi: 10.1002/advs.202516702

Figure Lengend Snippet: FBXL4 physically interacts with PFN1 to promote its degradation. (A) Bar plot showing Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of FBXL4‐binding genes. (B) The volcano plot highlights the top 13 genes exhibiting the most significant correlations with FBXL4. (C) The Venn diagram illustrates the overlap between 837 proteins identified by mass spectrometry and candidates prioritized by Spearman's correlation analysis, defining the putative substrate of FBXL4. (D) Scatter plots illustrating the correlation between FBXL4 and PFN1 in the single‐cell dataset ( GSE120064 ): A two‐tailed p ‐value < 0.05 denotes statistical significance, and an absolute Spearman correlation coefficient |R| ≥ 0.30 indicates a meaningful association. (E) IF staining showing the cellular distribution of FBXL4 and PFN1 in CMs. FBXL4 stained in green, PFN1 stained in red, and the nucleus stained in blue with DAPI. Scale bars: upper, 100 µm; lower, 10 µm. (F, G) Lysates from HEK293T cells transfected with Flag‐FBXL4 and HA‐PFN1 plasmids were immunoprecipitated with anti‐Flag/anti‐HA followed by WB with anti‐Flag (FBXL4) and anti‐HA (PFN1) ( n = 3). (H, I) Endogenous protein interactions were examined in cardiomyocyte lysates immunoprecipitated (IP) with anti‐rabbit IgG or anti‐FBXL4/PFN1 antibody, and analyzed by Western blot with antibodies to detect PFN1 and FBXL4 ( n = 3). (J) Schematic diagram for the function domain deletion mutant of PFN1 (left). Western blot of whole cell lysates and anti‐HA Co‐IP samples derived from HEK293T cells transfected with the indicated constructs (right), n = 3. (K) Representative immunoblotting analysis and quantification of PFN1, phosphorylated ERK1/2 (p‐ERK1/2 at Thr202/Tyr204), and ERK1/2 protein levels in FBXL4 fl/fl and FBXL4‐cKI mice hearts following TAC‐induced hypertrophy ( n = 6 hearts per group). Normalize phospho‐ERK1/2 to total ERK1/2. Normalize PFN1 and ERK1/2 to β‐actin. β‐actin was used as an internal control. (L) Representative immunoblotting analysis and quantification of PFN1, p‐ERK1/2, and ERK1/2 protein levels in FBXL4‐iCKO and Myh6‐Cre hearts ( n = 6 hearts per group). Normalize phospho‐ERK1/2 to total ERK1/2. Normalize PFN1 and ERK1/2 to GAPDH. GAPDH was used as an internal control. (M, N) Lysates from heart tissues of FBXL4‐cKI or FBXL4‐iCKO mice were immunoprecipitated with anti‐PFN1 antibody and blotted with total ubiquitin‐conjugated protein or PFN1 body, and quantification of the relative ubiquitinated PFN1 level ( n = 3). (O, P) NMCMs were infected with Ad‐GFP, Ad‐FBXL4, siRNA‐control or siRNA‐FBXL4, and then treated with Cycloheximide (CHX, 10 µ m ) for the indicated time periods. Representative western blot analysis of FBXL4 and PFN1 protein levels for each group (left), and quantification of PFN1 level (right; n = 4). n represents the number of independent samples per group. Data are shown as mean ± SD. ns . indicates no significance. * p < 0.05, ** p < 0.01, and *** p < 0.001. Statistical differences were assessed by one‐way ANOVA followed by Sidak post hoc multiple comparisons test (K) and unpaired t test with Welch's correction (L, M, N). Two‐way ANOVA with Bonferroni's post‐hoc test was used to determine the difference among the treatment groups and different time points (O, P).

Article Snippet: Supernatants were incubated overnight at 4°C with Protein A/G Magnetic Beads (HY‐K0202, MCE) pre‐bound to anti‐HA (51064‐2‐AP, Proteintech) or anti‐PFN1 (YN2843, Immunoway).

Techniques: Binding Assay, Mass Spectrometry, Single Cell, Two Tailed Test, Staining, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Co-Immunoprecipitation Assay, Derivative Assay, Construct, Control, Ubiquitin Proteomics, Infection

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: p53 signaling pathway was the major pathway regulated by Z-7 in p53 WT CRC cells. ( a ) Volcano plot of DEGs from RNA-seq of HCT116 cells treated with Z-7; ( b ) KEGG pathway enrichment analysis showed the DEGs were enriched in the p53 pathway and other cell cycle related pathways; ( c ) Heatmap of p53 pathway related genes expression in HCT116 treated with Z-7; ( d ) Heatmap of selected differentially expressed genes in both HCT116 and HT29 cells treated with Z-7; ( e-g ) qPCR analysis of p53 down-stream genes CDKN1A ( e ), MDM2 ( f ), and BBC3 ( g ) in Z-7 treated HCT116 cells; ( h ) KEGG pathway enrichment analysis showed the differential expressed proteins were enriched in the p53 signaling pathway and cell cycle related pathways; ( i ) Heatmap of p53 pathway related proteins expression in HCT116 treated with Z-7; ( j ) Cell cycle analysis of LoVo treated with different concentrations of Z-7 for 24 hours; ( k ) Quantification of Annexin V + cells percentage in LoVo treated with different concentrations of Z-7; ( l ) Western blot analysis of apoptotic markers in LoVo cells treated different concentrations of with Z-7 for 24 hours. CP, cellular process; EIP, encrionmental information processing; Meta, metabolism; OS, organismal systems. Statistical significance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Subsequently, diluted lysate mixed with 10 μL of protein A/G magnetic beads (HY-0202, MCE) and 1 μg of anti-p53 (2524S, CST) was incubated at 4 °C overnight.

Techniques: RNA Sequencing, Expressing, Cell Cycle Assay, Western Blot

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: Z-7 inhibited proliferation of p53 WT CRC cells through activation of the p53 signaling pathway. ( a-b ) Western blot analysis of p53 and p21 expression in HCT116 ( a ) and LoVo ( b ) after varied concentrations of Z-7 treatment; ( c-f ) qPCR analysis of TP53 ( c ) and down-stream genes CDKN1A ( d ), MDM2 ( e ), and BBC3 ( f ) in TP53 knockdown HCT116 cells after varied concentrations of Z-7 treatment; ( g ) Western blot analysis of apoptotic markers, p53, and p21 in TP53 knockdown HCT116 cells after varied concentrations of Z-7 treatment; ( h ) Quantification of Annexin V + cells percentage in TP53 knockdown HCT116 cells after varied concentrations of Z-7 treatment; ( i ) Cell cycle analysis of TP53 knockdown HCT116 treated with 2.5 µM Z-7. Statistical significance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Subsequently, diluted lysate mixed with 10 μL of protein A/G magnetic beads (HY-0202, MCE) and 1 μg of anti-p53 (2524S, CST) was incubated at 4 °C overnight.

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Cell Cycle Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: Z-7 restored p53 protein level through MDM2 mediated p53 ubiquitination by inhibiting E3 ligase function of MDM2. ( a ) Cyclohexamide (CHX) chasing assay showed Z-7 stabilizing p53 protein level; ( b ) Statistical data of two independent CHX chasing experiments was generated by using grayscale analysis in ImageJ; ( c ) Western blot analysis showed over expressing MDM2 can reverse the effects of Z-7 on p53 accumulation; ( d ) Western blot analysis showed Z-7 down regulated the MDM2 mediated ubiquitin level of p53; ( e ) The structure of Z-7 tagged with biotin label at the end of acetyl group (named Biotin-Z-7); ( f ) Competition assay showed the Z-7 physically interacted with MDM2. Whole cell lysis (WCL) of HEK-293T cells with over-expressed FLAG-MDM2 was incubated with or without 100 µM of Z-7 first, and then incubated with streptavidin beads coated with different concentrations of Biotin-Z-7, M for marker; ( g ) Co-IP showed Z-7 did not interrupt the MDM2-p53 interaction. Briefly, FLAG-MDM2 and Myc-p53 were co-transfected into 293T cells, followed by treatment with or without Z-7. Nutlin-3A served as a positive control. Samples were harvested after treatment, and subjected to immunoprecipitation using an anti-FLAG antibody. ( h ) Schematic overview of the truncated MDM2 fragments; ( i ) Pulldown assay showed the Z-7 bound to the RING domain of MDM2. WCL of HEK-293T cells with over-expressed truncated FLAG-MDM2 fragments were incubated with streptavidin beads coated with DMSO or 25 µM of Z-7; ( j ) Western blot analysis showed Z-7 inhibited MDM2 auto-ubiquitination. FLAG-MDM2 and HA-Ub were co-transfected into 293T cells following Z-7 treatment for 24 hours. Before sampls were harvested, samples were treated with or with 10 µM of MG-132 for 6 hours. Then samples were subjected to ubiquitin analysis and western blot analysis. Statistical significance is indicated as *, p < 0.05

Article Snippet: Subsequently, diluted lysate mixed with 10 μL of protein A/G magnetic beads (HY-0202, MCE) and 1 μg of anti-p53 (2524S, CST) was incubated at 4 °C overnight.

Techniques: Ubiquitin Proteomics, Generated, Western Blot, Expressing, Competitive Binding Assay, Lysis, Incubation, Marker, Co-Immunoprecipitation Assay, Transfection, Positive Control, Immunoprecipitation

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: A novel β-carboline alkaloid derivative targeting MDM2-p53 pathway suppresses colorectal cancer progression

doi: 10.1007/s13402-025-01111-3

Figure Lengend Snippet: Z-7 suppressed the growth of HCT116 xenograft model in mice. ( a ) Schematic overview and timeframe of the animal experiments. BALB/c nude mice were first inoculated subcutaneously with HCT116 cells. From day 6, control or Z-7 was administered via intratumorally injection. All mice were sacrificed on day 16 and the xenografts were excised for further analysis; ( b ) HCT116 xenograft tumor growth curves of intra-tumoral Z-7 administration in nude mice compared with control group; ( c ) The weight of HCT116 xenograft tumors in Z-7 treatment group compared with control group; ( d ) Representative image of HCT116 xenograft tumors with or without Z-7 treatment in nude mice; ( e ) Western blot analysis of p53 and p21 in HCT116 xenograft tumor treated with or without Z-7 in nude mice. 50 mg of sample was excised from each xenograft tumor, then homogenized and lysed with Laemmli lysis buffer, and subjected to immunoblotting analysis; ( f ) Immunohistochemistry analysis of p53 expression level in HCT116 xenograft tumor treated with or without Z-7; ( g ) Proposed model. Z-7 may inhibit MDM2 E3 ligase function, resulting downregulation of p53 ubiquitination level, thereby stabilizing p53 protein and activating relevant downstream proteins in the p53 signaling pathway, eventually leading to inhibit the colorectal cancer progression. Moreover, Z-7 also can increase the ROS level which also promotes cell cycle arrest and apoptosis in colorectal cancer. All results are shown as mean±S.E.M ( n = 5). * p < 0.05, ** p < 0.01，*** p < 0.001

Article Snippet: Subsequently, diluted lysate mixed with 10 μL of protein A/G magnetic beads (HY-0202, MCE) and 1 μg of anti-p53 (2524S, CST) was incubated at 4 °C overnight.

Techniques: Control, Injection, Western Blot, Lysis, Immunohistochemistry, Expressing, Ubiquitin Proteomics

Journal: Frontiers in Oncology

Article Title: RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport

doi: 10.3389/fonc.2021.698410

Figure Lengend Snippet: Expression of RanBP3 is up-regulated in CML. (A) The RanBP3 mRNA level was analyzed in database GSE33075, including 9 healthy individuals and 9 CML patients. After removing the outliers of each group, 7 samples of each group were used for expression analysis. (B) Bone marrow mononuclear cells separated from 3 normal controls and 4 CML patients were used to assess the mRNA levels of RanBP3. (C) The protein levels of RanBP3 were detected in BCR-ABL positive cells K562, K562/G01, KCL22 and BCR-ABL negative cells HL60, THP-1 and NB4. (E) The protein levels of RanBP3 in BCR-ABL transformed mouse cell lines and their corresponding control cells. (D, F) The mRNA levels of RanBP3 correspond to cells in (C) and (E) . (G, H) K562 and K562/G01 cells were treated with various concentrations of IM for 48 hours and the protein (G) and mRNA (H) levels of RanBP3 in these cells were detected. **P < 0.01, ***P < 0.001.

Article Snippet: 30μl protein A/G magnetic beads (MCE, USA) were incubated with anti-RanBP3 antibody (Cell Signaling Technology, USA) or isotype-matched control IgG at 4°C for 2h on a shaker, after that, magnetic beads were separated magnetically, washed by 1ml PBST for 4 times, and incubated another 2h with the proteins under the same condition.

Techniques: Expressing, Transformation Assay, Control

Journal: Frontiers in Oncology

Article Title: RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport

doi: 10.3389/fonc.2021.698410

Figure Lengend Snippet: Effect of RanBP3 on the proliferation of CML cells. K562 and K562/G01 cells were infected with the lentivirus-carrying shRanBP3 and control shRNAs for 72h and collected for following assays. (A, B) RanBP3 levels in shRanBP3 and control cells were measured by western blots and qRT-PCR. (C, D) Cell viability was detected by CCK-8 at indicated time-points. The size (E) and number (F) of colonies were measured in lentivirus infected K562 or K562/G01. The protein (G) and mRNA (H) levels of p21 were evaluated. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: 30μl protein A/G magnetic beads (MCE, USA) were incubated with anti-RanBP3 antibody (Cell Signaling Technology, USA) or isotype-matched control IgG at 4°C for 2h on a shaker, after that, magnetic beads were separated magnetically, washed by 1ml PBST for 4 times, and incubated another 2h with the proteins under the same condition.

Techniques: Infection, Control, Western Blot, Quantitative RT-PCR, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport

doi: 10.3389/fonc.2021.698410

Figure Lengend Snippet: Effect of RanBP3 on the apoptosis of CML cells. (A) Infected cells were labeled with Annexin V-PE and 7-AAD and examined by FCM. (B) The apoptosis results of three times in (A) were recorded and plotted as a bar graph. (C) DAPI staining was used to assess morphologic changes in apoptosis. The prominent apoptotic morphologies were indicated using white arrows. (D, E) Cleaved PARP and Caspase-3 were evaluated by western blot in lentivirus infected cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: 30μl protein A/G magnetic beads (MCE, USA) were incubated with anti-RanBP3 antibody (Cell Signaling Technology, USA) or isotype-matched control IgG at 4°C for 2h on a shaker, after that, magnetic beads were separated magnetically, washed by 1ml PBST for 4 times, and incubated another 2h with the proteins under the same condition.

Techniques: Infection, Labeling, Staining, Western Blot

Journal: Frontiers in Oncology

Article Title: RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport

doi: 10.3389/fonc.2021.698410

Figure Lengend Snippet: RanBP3 silencing increases the IM-induced apoptosis of CML cells. (A) The database GSE130404 contains 83 patients with early molecular response achieved (EMR achieved: BCR-ABL1 < 10% (IS) at 3 months) and 13 patients with early molecular response failure (EMR failure: BCR-ABL1 > 10% (IS) at 3 months). After excluding the outliers of each group, the P-value of RanBP3 expression of these two groups was calculated. (B, C) K562 (or K562/G01) cells and their derivatives were inoculated in 96-well culture plates (5000 cells/100μl per well), then treated with different concentrations of IM for 48h. The OD450nm value of each well was measured by CCK-8 and the cell survival rate was calculated. (D, E) Cells were infected with the lentivirus-carrying shRanBP3 and control shRNAs for 48h and followed by IM treatment for 48h (0.5μM IM for K562, 2μM IM for K562/G01). Apoptosis was examined by FCM. **P < 0.01, ***P < 0.001.

Article Snippet: 30μl protein A/G magnetic beads (MCE, USA) were incubated with anti-RanBP3 antibody (Cell Signaling Technology, USA) or isotype-matched control IgG at 4°C for 2h on a shaker, after that, magnetic beads were separated magnetically, washed by 1ml PBST for 4 times, and incubated another 2h with the proteins under the same condition.

Techniques: Expressing, CCK-8 Assay, Infection, Control

Journal: Frontiers in Oncology

Article Title: RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport

doi: 10.3389/fonc.2021.698410

Figure Lengend Snippet: RanBP3 silencing increases IM sensitivity in vivo . K562-shRanBP3 or K562-shNT cells were injected into the tail veins of NOD/SCID mice. 10 days after injection, 10mg/kg IM or control PBS was injected intraperitoneally for 14 days. (A) The total WBC counts in mice were determined. (B) The percentage of human CD45 + cells in BM cells of NOD/SCID mice was measured by FCM. (C) The images of livers and spleens were shown. The weights of livers (D) and spleens (E) were quantified. (F) Murine liver and spleen infiltration in 4 groups were analyzed by H&E, and bone marrow cells were checked with Wright’s stain. (G) Survival curves were drawn by GraphPad 8.0 and analyzed by Kaplan–Meier methods. *p< 0.05, **p< 0.01 and ***< 0.001.

Article Snippet: 30μl protein A/G magnetic beads (MCE, USA) were incubated with anti-RanBP3 antibody (Cell Signaling Technology, USA) or isotype-matched control IgG at 4°C for 2h on a shaker, after that, magnetic beads were separated magnetically, washed by 1ml PBST for 4 times, and incubated another 2h with the proteins under the same condition.

Techniques: In Vivo, Injection, Control, Staining

Journal: Frontiers in Oncology

Article Title: RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport

doi: 10.3389/fonc.2021.698410

Figure Lengend Snippet: RanBP3 regulates cell proliferation and apoptosis by mediating the nuclear transport of SMAD2/3 and ERK1/2. K562-shRanBP3 and K562-shNT were treated with DMSO or 10μM SB432542 for 8h. (A) The nuclear and cytosol proteins were extracted and used for the detection of SMAD2/3 by western blot. (B) Total proteins were obtained for the detection of p-SMAD2/3 and p21 levels. (C) Cell survival was determined by CCK-8 at the indicated time. The cytoplasmic and nuclear distribution of ERK1/2 was analyzed in K562-ShRanBP3, K562-shNT and K562 by western blot (D) and immunofluorescent (E) . (F) The expression of p(ser112)-BAD was determined in K562-ShRanBP3 and K562-shNT cells. (G) CO-IP assay was performed using the antibody anti-RanBP3 to examine the interaction of proteins in K562-ShRanBP3 and K562-shNT cells, and the RanBP3-bound proteins were determined by anti-ERK1/2, anti-CRM1, and anti-SMAD2/3 antibody. An anti-Rabbit IgG antibody was used as the negative control. (H) The working model for RanBP3 silencing-mediated proliferation and apoptosis in CML. RanBP3 is a downstream molecule of BCR-ABL and regulates SMAD2/3 and ERK1/2 nuclear export. After the knockdown of RanBP3, nuclear SMAD2/3 increased followed by the enhanced levels of p21, which inhibited cell proliferation, and the decreased cytoplasmic ERK1/2 levels relieved the antiapoptotic effect of p-BAD in CML. *p < 0.05, **p < 0.01 and ns means no significant.

Article Snippet: 30μl protein A/G magnetic beads (MCE, USA) were incubated with anti-RanBP3 antibody (Cell Signaling Technology, USA) or isotype-matched control IgG at 4°C for 2h on a shaker, after that, magnetic beads were separated magnetically, washed by 1ml PBST for 4 times, and incubated another 2h with the proteins under the same condition.

Techniques: Western Blot, CCK-8 Assay, Expressing, Co-Immunoprecipitation Assay, Negative Control, Knockdown

Journal: iScience

Article Title: PEA15 promotes osteosarcoma progression and cisplatin resistance by modulating autophagy through the FABP3-TNF signaling axis

doi: 10.1016/j.isci.2025.113695

Figure Lengend Snippet: Mechanistic insights into PEA15 downstream signaling (A) Heatmap of transcriptomic analysis in U2OS cells following PEA15 knockdown. (B) CCK-8 assay was adopted to assess cell proliferation after FABP3 knockdown in U2OS cells ( n = 3). (C) Colony formation assay in U2OS cells with FABP3 knockdown ( n = 3). (D and E) Migration and invasion assays in U2OS cells with FABP3 knockdown, along with the expression levels of related proteins ( n = 3). (F and G) Fluorescence imaging of autophagic activity and expression of autophagy-related proteins following FABP3 knockdown. (K–L) Flow cytometry analysis of apoptosis and expression of apoptosis-related markers after FABP3 knockdown ( n = 3). (M) KEGG pathway analysis of transcriptomic data from U2OS cells with PEA15 knockdown. (N) In U2OS cells with PEA15 knockdown, FABP3 was overexpressed, followed by the detection of ATG5 and LC3B protein expression levels.

Article Snippet: The remaining lysates were pre-cleared with Protein A/G magnetic beads (MCE, HY-K0202) for 1 h. Subsequently, the pre-cleared lysates were incubated overnight at 4°C with primary antibodies against PEA15 (Abcam, ab277626), FABP3 (CST, # 13368S), or with a control Normal Rabbit IgG (CST, #2729).

Techniques: Knockdown, CCK-8 Assay, Colony Assay, Migration, Expressing, Fluorescence, Imaging, Activity Assay, Flow Cytometry