Journal: Poultry Science

Article Title: RACK1 inhibits fowl adenovirus serotype 4 replication by targeting the viral protein Hexon for ubiquitin-proteasome degradation

doi: 10.1016/j.psj.2026.106627

Figure Lengend Snippet: The endogenous interaction between FAdV-4 Hexon and RACK1 in FAdV-4-infected LMH cells. ( A ) LMH cells were mock infected or infected with FAdV-4 at an MOI of 0.1. At 24 h post infection, cell lysates were prepared and immunoprecipitated with anti-Hexon mAb. Both the cell lysates and immunoprecipitates were examined by Western blotting. “Isotype” indicates the isotype control antibody (IgG2b) of anti- Hexon mAb. ( B ) LMH cells were mock infected or infected with FAdV-4 at an MOI of 0.1. At 24 h post infection, cells were fixed and probed with mouse anti-Hexon and rabbit anti-RACK1 antibodies, followed by incubation with FITC-conjugated goat anti-mouse antibodies (green) and TRITC-conjugated goat anti-rabbit antibodies (red). The nuclei were counterstained with DAPI (blue). The cell samples were observed with a confocal laser scanning microscope. Scale bar = 5 μm. (C) Liver tissues were collected from SPF chickens that infected with FAdV-4 or mock infection, followed by homogenization and preparation of tissue protein lysates. And then, the lysates were prepared and immunoprecipitated with anti-Hexon mAb. Both the lysates and immunoprecipitates were examined by Western blotting.

Article Snippet: Anti-c-Myc, anti-RACK1, anti-β-actin monoclonal antibodies and normal mouse IgG was purchased from Santa Cruz Biotechnology (USA).

Techniques: Infection, Immunoprecipitation, Western Blot, Control, Incubation, Laser-Scanning Microscopy, Homogenization

Journal: Poultry Science

Article Title: RACK1 inhibits fowl adenovirus serotype 4 replication by targeting the viral protein Hexon for ubiquitin-proteasome degradation

doi: 10.1016/j.psj.2026.106627

Figure Lengend Snippet: Both FAdV-4 infection and flag-hexon transfection have no effect on the expression of RACK1. ( A&B ) FAdV-4 infection had no effect on RACK1 expression. LMH cells were mock-infected or infected with FAdV-4 at an MOI of 0.1, 1, and 10. At 24 h post infection, cell lysates were harvested and examined by Western blotting using anti-Hexon, anti-RACK1 and anti-β-actin antibodies (A). LMH cells were mock-infected or infected with FAdV-4 at an MOI of 0.1 for different times. After infection, cell lysates were harvested and examined by Western blotting using anti-Hexon, anti-RACK1 and anti-β-actin antibodies (B). ( C&D ) Overexpression of Hexon had no effect on RACK1 expression. LMH cells were transfected with pRK5-flag or pRK5-flag-hexon at 0.2, 1, and 5 μg. At 24 h post infection, cell lysates were harvested and examined by Western blotting using anti-Hexon, anti-RACK1 and anti-β-actin antibodies (C). Endogenous β-actin expression was examined as an internal control and the relative protein levels of RACK1 in (C) were shown in panel D. Data are representative of three independent experiments and presented as means ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

Article Snippet: Anti-c-Myc, anti-RACK1, anti-β-actin monoclonal antibodies and normal mouse IgG was purchased from Santa Cruz Biotechnology (USA).

Techniques: Infection, Transfection, Expressing, Western Blot, Over Expression, Control

Journal: bioRxiv

Article Title: Subcellular proteomic profiling of human skeletal muscle reveals exercise-induced coordinated and compartment-specific protein remodeling

doi: 10.64898/2026.02.08.703653

Figure Lengend Snippet: a.) Heatmap of differentially expressed ribosomal proteins (adjusted P < 0.05) identified in the mitochondrial, nuclear, and cytosolic fractions. Colours indicate log□fold-change at each acute timepoint (MID, POST, 3H relative to PRE). b.) Scaled profile plots showing mean z-score changes of Receptor for Activated C Kinase 1 (RACK1) detected in the mitochondrial and nuclear fractions across PRE, MID, POST, and 3H. Subpanels illustrate trajectories for females (F), males (M) and combined sexes (F), obtained from the Subcell-EX Shiny app for visualising compartment- and sex-stratified comparisons. c.) Representative immunoblot images of RACK1 (5 µg total protein loaded per lane, including molecular weight marker and pooled control sample in the first lanes) across subcellular fractions (mitochondria, nucleus, cytosol) at PRE, MID, POST, and 3H. Lane colouring reflects fraction identity: mitochondria (peach), nucleus (blue), cytosol (green). A Stain-Free total protein image is shown below as a loading control. d) RACK1 subcellular protein abundance in response to exercise, determined using band densitometry and expressed in arbitrary units (a.u.). Quantified data are presented as mean ± SD for a representative subset of n = 10 participants (5 males, 5 females). Comparisons are shown for PRE, MID, POST, and 3H within each fraction (mitochondrial, nuclear, cytosolic). (*P ≤ 0.05; **P ≤ 0.001) relative to PRE, determined by pairwise t-tests with Bonferroni correction.

Article Snippet: Receptor for Activated C Kinase 1 (RACK1, Cell Signalling Technology, #5432 CST) was also interrogated via immunoblotting to validate MS results.

Techniques: Western Blot, Molecular Weight, Marker, Control, Staining, Quantitative Proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: SMYD3 synergises with RACK1 to promote colorectal cancer lung metastasis by recruiting SMAD3

doi: 10.1186/s12964-026-02687-5

Figure Lengend Snippet: SMYD3, RACK1 and SMAD3 interact with each other and the interaction between SMYD3 and SMAD3 depends on RACK1. A Silver staining results of proteins pulled down by Flag-SMYD3 in HCT116 cells. B The intersection veen plot of the immunoprecipitation and mass spectrometry analysis in colon cancer cell lines HCT15 and HCT116. C List of the candidate target proteins identified by the mass spectrometry. D Coimmunoprecipitation (Co-IP)-Western blot (WB) analysis of the SMYD3-RACK1 interaction in HCT116 cells transfected with Flag-tagged SMYD3 (Flag-SMYD3). E Co-IP-WB analysis of the SMYD3-RACK1-SMAD3 interaction in HCT116 cells transfected with Flag-tagged SMYD3 (Flag-SMYD3). F Immunoprecipitation experiments were performed to detect the endogenous interaction of SMYD3,SMAD3, RACK1 in HCT116 cells. G Immunofluorescence assay of endogenous SMYD3 and RACK1 in HCT116 cells. DAPI was used to counterstain the nucleus. H Immunofluorescence assay of endogenous SMYD3 and SMAD3 in HCT116 cells. DAPI was used to counterstain the nucleus. I Immunofluorescence assay of endogenous RACK1 and SMAD3 in HCT116 cells. DAPI was used to counterstain the nucleus. J Effects of RACK1 knockdown on SMYD3 interacted with SMAD3 in HCT116-LV-Flag-SMYD3 and RKO-LV-Flag-SMYD3 cells. K Effects of RACK1 knockdown on SMYD3 interacted with SMAD3 in HCT116-LV-His-SMAD3 and RKO-LV-His-SMAD3 cells

Article Snippet: Following treatment with EDTA antigen retrieval solution and goat serum, the slides were incubated overnight at 4 °C with primary antibodies, including SMYD3 (Abcam, #ab187149, 1:100), SMAD3 (Abclonal, #A19115, 1:200), RACK1 (CST, #5432, 1:200), and TSKU (Proteintech, #12,370–1-AP, 1:100).

Techniques: Silver Staining, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Transfection, Immunofluorescence, Knockdown

Journal: Cell Communication and Signaling : CCS

Article Title: SMYD3 synergises with RACK1 to promote colorectal cancer lung metastasis by recruiting SMAD3

doi: 10.1186/s12964-026-02687-5

Figure Lengend Snippet: RACK1 recruits SMAD3 to SMYD3 and promotes the transcriptional activation of the genes downstream of SMYD3-SMAD3. A Effect of RACK1 knockdown on TSKU, H3K4me3, p-SMAD3 protein expression in RKO cells. B Effect of RACK1 overexpression on TSKU, H3K4me3, p-SMAD3 protein expression in HCT8 cells. C Effect of RACK1 knockdown on TSKU mRNA expression in RKO cells. D Effect of RACK1 overexpression on TSKU mRNA expression in HCT8 cells. E ChIP-qPCR with anti-Flag antibody showed binding of SMYD3 to TSKU. F ChIP-qPCR with anti-His antibody showed binding of SMAD3 to TSKU Data were shown as mean ± SD. The data were analyzed by Two-way ANOVA. *** p < 0.001

Article Snippet: Following treatment with EDTA antigen retrieval solution and goat serum, the slides were incubated overnight at 4 °C with primary antibodies, including SMYD3 (Abcam, #ab187149, 1:100), SMAD3 (Abclonal, #A19115, 1:200), RACK1 (CST, #5432, 1:200), and TSKU (Proteintech, #12,370–1-AP, 1:100).

Techniques: Activation Assay, Knockdown, Expressing, Over Expression, ChIP-qPCR, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SMYD3 synergises with RACK1 to promote colorectal cancer lung metastasis by recruiting SMAD3

doi: 10.1186/s12964-026-02687-5

Figure Lengend Snippet: SMYD3-SMAD3 promotes colon cancer cell metastasis in vitro and in vivo dependent on RACK1. A Wound healing assay shows the effect of RACK1 knockdown on vector-, SMYD3-overexpressing and SMAD3-overexpressing RKO and HCT116 cell metastases. B Transwell assay shows the effect of RACK1 knockdown on vector-, SMYD3-overexpressing and SMAD3-overexpressing RKO and HCT116 cell metastases. C Wound healing assay shows the effect of RACK1 overexpression on vector-, SMYD3- knockdown and SMAD3- knockdown RKO and HCT8 cell metastases. D Transwell assay shows the effect of RACK1 overexpression on vector-, SMYD3- knockdown and SMAD3- knockdown RKO and HCT8 cell metastases. E The schematic diagram of tail vein lung metastasis model construction. F Representative living images of mice injected with HCT116 transfected by indicated lentivirus into tail vein. The lentivirus was Luci-labelled and therefore stably transfected HCT116 cell lines had in vivo luciferase activity. G Statistical analysis of luciferase bioluminescence intensity ( n = 5). H Statistical analysis of the number of pulmonary metastases of each group ( n = 5). I Representative images of metastases in murine lung of each group and H&E staining of pulmonary tissue sections; The black arrow indicated the metastasis. For A,B,C and D, significance was determined with the Two-way ANOVA. For G and H, significance was determined with the student unpaired t test. ns, not significant, p > 0.05; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. Errorbars, ± SD

Article Snippet: Following treatment with EDTA antigen retrieval solution and goat serum, the slides were incubated overnight at 4 °C with primary antibodies, including SMYD3 (Abcam, #ab187149, 1:100), SMAD3 (Abclonal, #A19115, 1:200), RACK1 (CST, #5432, 1:200), and TSKU (Proteintech, #12,370–1-AP, 1:100).

Techniques: In Vitro, In Vivo, Wound Healing Assay, Knockdown, Plasmid Preparation, Transwell Assay, Over Expression, Injection, Transfection, Stable Transfection, Luciferase, Activity Assay, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: SMYD3 synergises with RACK1 to promote colorectal cancer lung metastasis by recruiting SMAD3

doi: 10.1186/s12964-026-02687-5

Figure Lengend Snippet: TSKU is highly expressed in colorectal cancer and associated with poor prognosis. A Box plot shows TSKU expression in Para-carcinoma and colorectal cancer tissues (Tumor) from TNMplot database. B mRNA expression of TSKU in colon cancer tissues (Tumor) and corresponding paracancerous tissue (P) ( n = 8). C Western blot analysis of TSKU expression in CRC tissues (T) and corresponding paracancerous tissue (P) ( n = 8). D Immunohistochemical (IHC) staining of TSKU expression levels in tumor and normal tissues.A total of 76 pairs of tumor and normal tissues were analysed. E Kaplan–Meier estimates of OS of patients with strong positive TSKU expression vs those with weak positive TSKU expression. F Representative images of immunohistochemical staining of SMYD3、SMAD3、 RACK1 and TSKU of colon cancer specimens ( n = 76). G TSKU expression correlates with SMYD3 levels in tissue microarray of colorectal cancer samples. Protein levels of TSKU and SMYD3 were quantified in colon cancer specimens. H TSKU expression correlates with SMAD3 levels in tissue microarray of colorectal cancer samples. Protein levels of TSKU and SMAD3 were quantified in colon cancer specimens. I TSKU expression correlates with RACK1 levels in tissue microarray of colorectal cancer samples. Protein levels of TSKU and RACK1 were quantified in colon cancer specimens. For B, significance was determined with the student unpaired t test. For E, significance was determined with Log–rank (Mantel–Cox) test. ns, not significant, p > 0.05; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. Errorbars, ± SD

Article Snippet: Following treatment with EDTA antigen retrieval solution and goat serum, the slides were incubated overnight at 4 °C with primary antibodies, including SMYD3 (Abcam, #ab187149, 1:100), SMAD3 (Abclonal, #A19115, 1:200), RACK1 (CST, #5432, 1:200), and TSKU (Proteintech, #12,370–1-AP, 1:100).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Immunohistochemistry, Staining, Microarray