anti pakt  (Proteintech)

Journal: bioRxiv

Article Title: Structural semantic evolutionary distance (SSED) unifies the selection of cancer driver genes across macroevolution and tumorigenesis

doi: 10.64898/2025.12.17.694808

Figure Lengend Snippet: A: Amino acid residue contact matrix of the wild-type KRAS protein. B: Differences in the residue contact matrix and superimposed 3D structural (predicted by ESM3) comparisons between the wild-type KRAS protein and four mutants (R135T, R135L, R135N, and R135P). C: Clonal competition assay assessing the proliferative advantage of mutants with different SSED values. Quantitative analysis (mean±SEM, n = 3, one-way ANOVA) of the proportion of GFP-positive cells (representing the wild-type population) after co-culture with mutants in BEAS-2B and hTERT-HPNE cell lines under normal (10% FBS) and low-nutrient stress (0.5% FBS) conditions. D: Representative fluorescence images of the co-culture (scale bar: 200μm). E, F: Transwell invasion assay evaluating the invasive capacity of different mutants. E: Representative images of invaded cells. F: Quantitative analysis of invaded cells (mean±SEM, n = 3, one-way ANOVA). G, H: Western blot analysis of downstream signaling pathway activation. G: Representative bands for p-AKT, p-ERK1/2, and β-actin. H: Quantitative analysis of pAKT and pERK1/2 (normalized to total protein and β-actin, mean±SEM, n = 3, one-way ANOVA). WT: wile-type, ns: No significant difference, *: P<0.05, **: P<0.005, ***: P<0.0005.

Article Snippet: Membranes were incubated with antibodies against pERK (Thr202/Tyr204, Proteintech, 28733-1-AP), pAKT (Ser473, Proteintech, 66444-1-Ig), and β-actin.

Techniques: Residue, Competitive Binding Assay, Co-Culture Assay, Fluorescence, Transwell Invasion Assay, Western Blot, Activation Assay

Journal: Cancer Research

Article Title: tRF-21 LeuTAA Promotes Oxidative Stress by Altering Glutathione Metabolic Enzymes to Support Prostate Cancer Progression

doi: 10.1158/0008-5472.CAN-25-0273

Figure Lengend Snippet: The Cys-Gly peptidase activity of LAP3 promoted ROS elevation and AKT activation by enhancing GSH degradation. A, si- LAP3 , the LAP3 dipeptidase activity inhibitor bestatin, and the tRF-21 inhibitor all significantly suppressed LAP3 and AKT enzyme activities and reduced p-mTOR S2481 and pAKT S473 levels, whereas artificial LAP3 overexpression with pc-LAP3 and treatment with tRF-21 mimics significantly increased LAP3 and AKT enzyme activities, as well as p-mTOR S2481 and pAKT S473 levels. B, A significant increase in intracellular GSH levels and a decrease in GSH degradation products Cys and Gly (resulting from Cys-Gly dipeptidase activity) were induced by si- LAP3 , the LAP3 dipeptidase activity inhibitor bestatin, and the tRF-21 inhibitor, whereas artificial LAP3 overexpression with pc- LAP3 and treatment with tRF-21 mimics showed effects to the contrary. C and D, A significant decrease in intracellular ROS ( C ) and an increase in TAC ( D ) were significantly induced by si- LAP3 , bestatin, and the tRF-21 inhibitor, whereas artificial LAP3 overexpression with pc- LAP3 and treatment with tRF-21 mimics showed effects to the contrary Scale bars, 100 μm. ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). E, Levels of AKT downstream targets pPRAS40 T246 , pGSK3β S9 , and pFOXO1 T24 were significantly reduced by si-LAP3, bestatin, and the tRF-21 inhibitor and increased by artificial LAP3 overexpression or tRF-21 mimics. F, Schematic summary of LAP3-mediated GSH degradation and the consequent increase in intracellular ROS levels, which resulted in AKT activation in prostate cancer. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: The anti-LAP3 (rabbit polyclonal, 1:100, A24554, ABclonal, RRID:AB_3665785), anti–pAKT S473 (mouse monoclonal, 1:80, 66444-1-Ig, Proteintech, RRID:AB2782958), and anti–Ki-67 (rabbit polyclonal, 1:100, ab16667, Abcam, RRID:AB_302459) antibodies were used for IHC.

Techniques: Activity Assay, Activation Assay, Over Expression

Journal: Cancer Research

Article Title: tRF-21 LeuTAA Promotes Oxidative Stress by Altering Glutathione Metabolic Enzymes to Support Prostate Cancer Progression

doi: 10.1158/0008-5472.CAN-25-0273

Figure Lengend Snippet: tRF-21 and LAP3 promoted prostate cancer tumor growth/progression and were associated with an unfavorable prognosis. A, Nude mice prostate cancer subcutaneous xenografts at day 21 after inoculation of DU145 cells with LAP3 stable knockdown (sh- LAP3 ) or sh-con. B, sh - LAP3 significantly reduced tumor growth (left), tumor size (middle), and weight (mean ± SD, n = 5 per group; right). C, Hematoxylin and eosin (H&E) and IHC of xenograft tumor tissues demonstrated that sh- LAP3 significantly decreased LAP3 expression, pAKT S473 level, and Ki-67 index. Scale bars, 50 μm. D, DU145 cell spheroid growth was repressed by the tRF-21 inhibitor, which was restored by cotreatment with tRF-21 mimics (left), whereas cotransfection of si- LAP3 reduced the effects of tRF-21 mimics (right). Scale bars, 50 μm. E, Tumor initiation assays demonstrated that sh - LAP3 markedly impeded xenograft initiation. F, sh- LAP3 significantly increased the chemosensitivity of DU145 cells to DOC, similar to the chemosensitizing effects of antioxidant VitE. G, RNA-ISH showed tRF-21 was overexpressed in prostate cancer (PCa) tissues in comparison with BPT. The violin plots (right) represent relative nuclear tRF-21(N + ) and cytoplasmic tRF-21(C + ) densities. Scale bars, 100 μm. H, Kaplan–Meier analysis with log-rank test showed tRF-21(N + ) and tRF-21(C + ) were adverse prognostic factors for PFS and OS of prostate cancer. I, Multivariate Cox proportional hazards model analysis of prostate cancer ( n = 91) showed that stage 4, tRF-21(N + ), and Gleason score ≥8 were independent risk factors for PFS, whereas stage 4 and tRF-21(N + ) were independent risk factors for OS (all P < 0.05). J, Relative expression level of LAP3 mRNA in prostate cancer was higher than that in normal tissue samples from the TCGA-PRAD dataset. K, PFS (left) and OS (right) in the TCGA-PRAD cohort showed higher LAP3 levels predicted poorer prognosis. Cutoffs were based on fragments per kilobase of transcripts per million mapped reads (FPKM). CI, confidence interval. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: The anti-LAP3 (rabbit polyclonal, 1:100, A24554, ABclonal, RRID:AB_3665785), anti–pAKT S473 (mouse monoclonal, 1:80, 66444-1-Ig, Proteintech, RRID:AB2782958), and anti–Ki-67 (rabbit polyclonal, 1:100, ab16667, Abcam, RRID:AB_302459) antibodies were used for IHC.

Techniques: Knockdown, Expressing, Cotransfection, Comparison