Journal: iScience

Article Title: MYCT1 alters the glycogen shunt by regulating selective translation of RACK1-mediated enzymes

doi: 10.1016/j.isci.2022.103955

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-RACK1 , Cell Signaling Technology , Cat# 5432; RRID: AB_10705522.

Techniques: Virus, Cloning, Recombinant, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, cDNA Synthesis, Mass Spectrometry, Negative Control, Software

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Immunohistochemical analysis shows co-aggregation of RACK1 with FUS in the cytoplasm ( arrows ) in spinal cord sections of a fALS-FUS-R521C case, in contrast to normal nuclear expression of FUS in control (Ctrl). Nuclei stained with DAPI in merged images. Scale bar: 10 μm

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Immunocytochemical analysis demonstrates expression of HA-tagged TDP-43 ΔNLS ( a , bottom row ) or FUS ΔNLS mutants, R495x- and P525L-FUS ( b , middle & bottom rows ), but not WT TDP-43 ( a , top row ) or WT-FUS ( b , top row ), which are primarily localized to the nucleus, is associated with co-aggregation of endogenous RACK1 in HEK293T cells. Nuclei stained with DAPI in merged images. Scale bars: 20 μm

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Expressing, Staining

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Immunocytochemical analysis demonstrates reactivity of RACK1 misfolding specific antibody “RACK1 mis ” with RACK1 in cytoplasmic aggregates of HA-tagged TDP-43 ΔNLS ( middle row ) or R495x-FUS ( bottom row ) transfected HEK293T cells but not with diffuse, non-aggregated RACK1 in the cytoplasm (stained by a Pan RACK1 antibody). RACK1 mis shows no reactivity with endogenous, physiological RACK1 in un-transfected (UT, top row ) cells. Nuclei stained with DAPI in merged images. Scale bars: 10 μm

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Transfection, Staining

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: RACK1 knockdown alleviates aggregation and global translational suppression by TDP-43 ΔNLS and WT over-expression in HEK293T cells. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control, expression of HA-tagged TDP-43 ΔNLS and to a lesser extent over-expression of WT TDP-43, induces a significant reduction in puromycin (PMY) incorporation. b RACK1 siRNA KD alleviates global translational levels in both cases as determined by quantification of PMY band intensities normalized to loading control β-actin. c SUnSET-ICC shows inhibition of PMY incorporation by TDP-43 ΔNLS (HA) expression preferably occurs in cells containing distinctive cytoplasmic aggregates ( asterisks, top row ). In cells where TDP-43 ΔNLS displays a filamentary expression pattern ( arrows, bottom row ), PMY incorporation is comparable to neighbouring HA negative UT cells. d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression ( top row ) or predominantly nuclear localization ( bottom row ) of TDP-43 ΔNLS in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics : Ordinary one-way ANOVA Tukey multiple comparisons. n = 4 * p < 0.05; *** p < 0.001; **** p < 0.0001. Scale bars: 20 μm

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Over Expression, Western Blot, Expressing, Inhibition, Transfection, Staining

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: RACK1 knockdown alleviates aggregation and restores global translational suppression by FUS ΔNLS mutants. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control empty vector (EV) transfected cells, expression of HA-tagged FUS ΔNLS mutants, R495x- and P525L-FUS, and to a lesser extent over-expression of WT FUS, induces a significant reduction in puromycin (PMY) incorporation. b siRNA KD alleviates global translational levels in all cases as determined by quantification of PMY band intensities normalized to loading control α-tubulin. c SUnSET-ICC demonstrates HA-tagged FUS ΔNLS mutants, R495x- and P525L-FUS, cause aggregation and completely inhibit PMY incorporation in transfected cells ( asterisks ). d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression or predominantly nuclear localization of HA-tagged FUS ΔNLS mutants, R495x- and P525L-FUS, in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics : Student’s t -test, unpaired, two-tailed. * p < 0.05 and ** p < 0.005. n = 5 Error bars: SEM. Scale bars: 20 μm

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Western Blot, Plasmid Preparation, Transfection, Expressing, Over Expression, Staining, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Immunocytochemical analysis shows co-aggregation of HA-tagged TDP-43 ΔNLS ( a , b ) or R495x-FUS ( c , d ) with RACK1 and eukaryotic 40S (Rps6) and 60S (RPL14) ribosome subunits ( arrows ). In neighbouring HA-negative UT cells, RACK1 is diffusely localized in the cytoplasm. Nuclei stained with DAPI in merged images. Scale bars: 10 μm

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Staining

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: RACK1 knockdown not only reduces cytoplasmic aggregation but also increases nuclear localization of TDP-43 ΔNLS in HEK293T cells. a Representative images showing that in contrast to control cells where distinctive aggregates of HA-tagged TDP-43 ΔNLS are associated with RACK1 co-aggregation in the cytoplasm ( arrows ), RACK1 siRNA KD not only alleviates cytoplasmic aggregation ( white arrowheads ) but also leads to nuclear localization ( yellow arrowheads ) of TDP-43 ΔNLS in a sub-population of transfected cells. Nuclei stained with DAPI in merged images. Scale bar: 10 μm. b Quantification of the average aggregate size in each individual TDP-43 ΔNLS transfected cells shows a decrease following RACK1 KD. c Quantification of TDP-43 ΔNLS nuclear localization from 3 biological repeats shows an increase as a result of RACK1 KD. d Western blot analysis confirms the quality of nucleocytoplasmic fractionation using nuclear and cytosolic protein markers, lamin B1 and β-Actin, respectively ( top ). TDP-43 ΔNLS (HA) or total TDP-43 (pan TDP-43) from either fraction was normalized to β-actin or lamin B1. Quantification by densitometry shows that RACK1 KD decreases the cytoplasmic to nuclear ratio, fragmentation ( red arrows , observed in 3 independent experiments), and phosphorylation of TDP-43 ΔNLS (quantitated in one experiment). Statistics : Student’s t -test unpaired two-tailed. Error bars: SEM

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Transfection, Staining, Western Blot, Fractionation, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: RACK1 knockdown inhibits intercellular transmission of TDP-43 ΔNLS in HEK293T cells. a Schematic illustration of the intercellular transmission procedure. Representative western blot (WB) b and densitometric quantification show reduced total HA-tagged TDP-43 ΔNLS transmitted to naïve recipient cells c as well as a reduction in the recipient/donor ratio of TDP-43 ΔNLS upon RACK1 KD. Statistics : Student’s t -test, unpaired two-tailed. ** p < 0.005; **** p < 0.0001. n = 5

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Transmission Assay, Western Blot, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: RACK1 knockdown significantly reduces hTDP-43-induced neuronal death and dysfunction in transgenic D. melanogaster. a Schematic illustration for the construction of transgenic D. melanogaster lines specifically targeting retinal (GMR-driven) or motor (D42-driven) neurons. b Representative images of fly eyes. Expression of hTDP-43 WT (A–D) or hTDP-43 Q331K (E–H) in retinal neurons cause neuronal death in both males and females, detected as darkened ommatidia. RACK1-RNAi reduces the appearance of dead ommatidia in all four conditions (B, D, F, H), compared to mCherry-RNAi control (A, C, E, G). Control flies with no dead ommatidia include those expressing RACK1-RNAi alone (I), and those harbouring unexpressed (undriven) transgenes (J–L). Scale bar: 250 mm. c Quantification of retinal neuronal death. Mann–Whitney test indicates a significant reduction in ommatidia death upon RACK1-RNAi KD in both males and females expressing either hTDP-43 WT or hTDP-43 Q331K . 21–33 flies were scored per condition. Statistics: Mann–Whitney test, * p < 0.05, *** p < 0.001. **** p < 0.0001. d Histological analysis of retina from flies expressing hTDP-43 WT (A, B) or hTDP-43 Q331K (C, D) in conjunction with control mCherry - RNAi (A, C) or RACK1-RNAi (B, D) show preservation of retinal architecture by RACK1 KD. Scale bar: 50 mm. e Both male and female hTDP-43 WT transgenic flies show reduced climbing ability compared to mCherry-RNAi controls, which was significantly improved by RACK1-RNAi. Statistics: 2-way ANOVA. ** p < 0.01, **** p < 0.0001. 60–100 flies were scored per condition. Error bars: SD

Article Snippet: The following primary antibodies were incubated for 1 h at RT or overnight at 4 °C: rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1,000), chicken polyclonal anti-HA (Abcam, ab9111, 1:10,000), rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1,000), mouse monoclonal anti-RACK1 (BD Biosciences, San Jose, CA, USA, 610178, 1:500), rabbit monoclonal RACK1 mis (ProMIS Neurosciences, see below, 1 μg/ml), rabbit monoclonal anti-RpS6 (Cell Signaling, Danvers, MA, USA, 5G10, 1:100), rabbit polyclonal anti-RPL14 (Bethyl Laboratories, Montgomery, TX, USA, A305-052, 1:1,000), mouse monoclonal anti-puromycin (ThermoFisher, clone 12D10, 1:1,000), rabbit monoclonal anti-Flag (Cell Signaling, 2368S, 1:2,000).

Techniques: Transgenic Assay, Expressing, MANN-WHITNEY, Preserving

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Nsp1 N-terminal and central domain mutants are defective for ribosome and mRNA binding (A) HEK293T cells were transfected with plasmids expressing WT or the indicated mutant 3xFLAG-Halo-tagged nsp1. Nsp1 was immunoprecipitated (IP) using ⍺-FLAG beads and coIP of ribosomal proteins RACK1, RPS2, RPS3, and RPS24 was monitored by western blotting, with vinculin serving as a loading control. Input lanes contain 1/10 of the amount of protein used for the IPs. (B) Equilibrium binding measurements of fluorescently labeled WT (blue), R124A,K125A (red), and R99A (green) nsp1 to purified ribosomes. Data represent a total of 3 biological replicates. (C) HEK293T cells were co-transfected with HBB-nLuc and either a control plasmid or the indicated 3xFLAG-Halo-tagged nsp1 constructs. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating RNAs were extracted and nLuc mRNA was quantified by qRT-PCR. The mRNA values were then normalized to the values obtained from the empty vector control. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (D) HEK293T cells were co-transfected with a 3xFLAG-Halo-tagged nsp1 plasmid or empty vector control, together with a plasmid expressing either GFP with a 5′ stem loop (GFP+SL) or a control GFP lacking the stem loop (GFP). Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating GFP+SL or GFP mRNAs were quantified by qRT-PCR. The mRNA values were then normalized to those obtained from the empty vector control. The bars represent the mean value of the replicates and error bars represent standard deviation. (E) The levels of GFP+SL and GFP mRNA present in the input samples from (D) were quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 (empty vector control) set to 1. Each dot represents an independent experiment. ∗∗ p ≤ 0.01; unpaired t test. See also , , and . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), mouse anti-FLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05), and HRP goat anti-rabbit IgG (1:10,000 SouthernBiotech 4030-05).

Techniques: Binding Assay, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Labeling, Purification, Plasmid Preparation, Construct, Quantitative RT-PCR, Standard Deviation

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), mouse anti-FLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05), and HRP goat anti-rabbit IgG (1:10,000 SouthernBiotech 4030-05).

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Transfection, Luciferase, SYBR Green Assay, Primer Extension Assay, Clone Assay, Software

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: Protein A/G magnetic beads were incubated with anti-Flag (CST, #14793S), anti-SMAD3 antibody (CST, #9523S, 1:100), anti-RACK1 antibody (CST, #5432S, 1:100), anti-His antibody (Proteintech, #66,005–1-Ig, 1:1000), or IgG (Beyotime, #A7058) for 4 h at 4 °C on a rotary shaker.

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: Protein A/G magnetic beads were incubated with anti-Flag (CST, #14793S), anti-SMAD3 antibody (CST, #9523S, 1:100), anti-RACK1 antibody (CST, #5432S, 1:100), anti-His antibody (Proteintech, #66,005–1-Ig, 1:1000), or IgG (Beyotime, #A7058) for 4 h at 4 °C on a rotary shaker.

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: Protein A/G magnetic beads were incubated with anti-Flag (CST, #14793S), anti-SMAD3 antibody (CST, #9523S, 1:100), anti-RACK1 antibody (CST, #5432S, 1:100), anti-His antibody (Proteintech, #66,005–1-Ig, 1:1000), or IgG (Beyotime, #A7058) for 4 h at 4 °C on a rotary shaker.

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: Protein A/G magnetic beads were incubated with anti-Flag (CST, #14793S), anti-SMAD3 antibody (CST, #9523S, 1:100), anti-RACK1 antibody (CST, #5432S, 1:100), anti-His antibody (Proteintech, #66,005–1-Ig, 1:1000), or IgG (Beyotime, #A7058) for 4 h at 4 °C on a rotary shaker.

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