Journal: Transboundary and Emerging Diseases

Article Title: RACK1 Associates With STING to Promote Type I Interferon Activation and Inhibit Pseudorabies Virus Infection

doi: 10.1155/tbed/9584967

Figure Lengend Snippet: The mechanism model in this study. Our model depicts the proposed mechanism by which RACK1 restricts PRV infection. RACK1 can bind to STING, promote the aggregation of STING around the Golgi apparatus, and thereby activate the IRF3–dependent IFN‐I signaling pathway to trigger an antiviral response. The figure was created with BioGDP.com .

Article Snippet: To assay for overexpression of RACK1, PK‐15 cells were seeded onto 12‐well plates (~2 × 10 5 cells per well) and then treated with the transfection complex containing TransIT‐X2 Transfection Reagent (Mirus, USA) and the overexpression vector of RACK1 (pcDNA3.1‐RACK1/p3 × FLAG‐CMV‐RACK1) or empty plasmid (pcDNA3.1(+)/p3 × FLAG‐CMV) for 48 h. Subsequently, the PK‐15 cells were infected with PRV JS‐2012 or stimulated with 2 ′ ,3 ′ ‐cGAMP (MedChemExpress, China) for the indicated time.

Techniques: Infection

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

Journal: PLoS ONE

Article Title: Nuclear Import of Transcription Factor BR-C Is Mediated by Its Interaction with RACK1

doi: 10.1371/journal.pone.0109111

Figure Lengend Snippet: (A) Yeast two-hybrid screen of BR-C-interacting proteins from a silkworm silk gland cDNA library using the BTB domain of BR-C as bait. A total of 54 positive clones were found to grow on selective medium. A negative control consisting of pGBKT7-Lam with pGADT7-T and a positive control consisting of pGBKT7-53 with pGADT7-T were used to measure the efficiency of the system. (B) Yeast two-hybrid confirmation of the interaction between BR-C and its novel interacting partner RACK1. cDNA encoding full-length BR-C, the BTB domain or partial BR-C lacking the BTB domain was cloned into the pGBKT7 vector. The full-length cDNA sequence of RACK1 gene was inserted into the pGADT7 vector. The yeast two-hybrid experiment was performed to test the interaction between BR-C and RACK1.

Article Snippet: The proteins in the supernatants were immunoblotted with anti-RACK1 antibody (Cell signaling technology; 1∶100).

Techniques: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Negative Control, Positive Control, Plasmid Preparation, Sequencing

Journal: PLoS ONE

Article Title: Nuclear Import of Transcription Factor BR-C Is Mediated by Its Interaction with RACK1

doi: 10.1371/journal.pone.0109111

Figure Lengend Snippet: (A) Interaction between BR-C and RACK1 by far-western blotting. GST, GST-tagged RACK1 (GST-RACK1), and SUMO-tagged BR-C (SUMO-BR-C) were expressed and purified from prokaryotic cells. The purified proteins were separated on 12% SDS-PAGE gels and transferred onto PVDF membranes, which were incubated with SUMO-BR-C or GST proteins at 4°C overnight, and analyzed by western blotting using anti-BR-C or anti-GST antibodies. (B-C) Interaction between BR-C and RACK1 by GST pull-down assay. Purified SUMO-BR-C was incubated with purified GST-RACK1 and examined by immunoblotting using an anti-BR-C antibody (B). Precleared BmN4 cell lysates containing endogenous RACK1 were incubated with purified GST-BR-C at 4°C for 6 h, and were separated via 12% SDS-PAGE gel and visualized by immunoblotting using an anti-RACK1 antibody (C).

Article Snippet: The proteins in the supernatants were immunoblotted with anti-RACK1 antibody (Cell signaling technology; 1∶100).

Techniques: Far Western Blot, Purification, SDS Page, Incubation, Western Blot, Pull Down Assay

Journal: PLoS ONE

Article Title: Nuclear Import of Transcription Factor BR-C Is Mediated by Its Interaction with RACK1

doi: 10.1371/journal.pone.0109111

Figure Lengend Snippet: RACK1 fused with Venus (Venus-RACK1) and BR-C fused with Venus (Venus-BR-C) were separately transfected into BmN4 cells. Two days post transfection, the cells were fixed and observed using confocal microscopy. The DNA was stained with DAPI.

Article Snippet: The proteins in the supernatants were immunoblotted with anti-RACK1 antibody (Cell signaling technology; 1∶100).

Techniques: Transfection, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Nuclear Import of Transcription Factor BR-C Is Mediated by Its Interaction with RACK1

doi: 10.1371/journal.pone.0109111

Figure Lengend Snippet: (A) BTB domain deletion affects the nuclear localization of exogenously expressed BR-C in BmN4 cells. Constructs expressing full-length BR-C and partial BR-C lacking the BTB domain were fused to DsRed and transfected into BmN4 cells, and the cells were observed by confocal microscopy two days after transfection. (B) RNAi against endogenous RACK1 gene inhibits the nuclear import of exogenously expressed BR-C in BmN4-SID1 cells. Double-stranded RNA (dsRNA) targeted the RAC K1 gene or the control EGFP gene was added to the culture medium separately for BmN4-SID1 cells, and five days later, the plasmid expressing full-length BR-C fused to DsRed was transfected into BmN4-SID1 cells. Confocal microscopy analysis was performed two days after transfection. Treatment with dsRNA against enhanced green fluorescent protein ( EGFP ) gene was used as a control. DAPI was used to stain the nuclear DNA of the cells.

Article Snippet: The proteins in the supernatants were immunoblotted with anti-RACK1 antibody (Cell signaling technology; 1∶100).

Techniques: Construct, Expressing, Transfection, Confocal Microscopy, Control, Plasmid Preparation, Staining

Journal: PLoS ONE

Article Title: Nuclear Import of Transcription Factor BR-C Is Mediated by Its Interaction with RACK1

doi: 10.1371/journal.pone.0109111

Figure Lengend Snippet: The scaffolding protein RACK1 recruits and activates PKC in the cytoplasm. After being translated in the cytoplasm, BR-C interacts with RACK1 and is phosphorylated by RACK1-anchored PKC at amino acid residues Ser373 and Thr406. Phosphorylated BR-C then translocates into the nucleus and subsequently activates the transcription of its target genes.

Article Snippet: The proteins in the supernatants were immunoblotted with anti-RACK1 antibody (Cell signaling technology; 1∶100).

Techniques: Scaffolding

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Mass spectrometry analysis of NLRP3, RACK1, and NEK7 peptides after purification of NLRP3 complexes.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Mass Spectrometry, Purification

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A–C) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). The cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h), ATP (5 mM, 30 min), or LPS (200 ng mL−1, 4 h) plus ATP (5 mM, 30 min). (A) Supernatants (Sup.) and cell lysates (Lys.) were analyzed by immunoblotting with the indicated antibodies. (B and C) IL-1β and TNF-α release in supernatants was analyzed by ELISA.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A–E) Macrophages (NLRP3 R258W) were treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h). Cell lysates (Lys.) and culture supernatants (Sup.) were immunoblotted with indicated antibodies (A and D). IL-1β (B), TNF-α (C), and LDH(E) in culture supernatants of macrophages were analyzed. Error bars denote SD of triplicate wells. Results are representative of three independent experiments. Unpaired two-tailed Student’s t test, *p < 0.05.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Two Tailed Test

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were primed with LPS (200 ng mL−1, 4 h) and then stimulated with ATP (5 mM, 30 min), nigericin (5 μM, 1 h), or Salmonella (MOI = 10, 1 h). Representative images of ASC specks in macrophages treated with indicated stimuli. Scale bars, 5 μm.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques:

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Tagged NLRP3 (NLRP3-SFP) was co-expressed with HA-tagged wild-type RACK1 or mutant RACK1 (R36D/K38E, defective in ribosome binding) in HEK293T cells. Cell lysates were pulled down with streptavidin beads and analyzed by immunoblotting.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Mutagenesis, Binding Assay, Western Blot

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Macrophages were infected with lentivirus expressing control shRNA (shControl) or RACK1 shRNA (shRACK1). After puromycin selection, transduced macrophages were primed with LPS (200 ng mL−1, 4 h) and then stimulated with PBS (control), ATP (5 mM, 30 min), or nigericin (5 μM, 1 h). NLRP3 inflammasome assembly was analyzed by blue native PAGE and immunoblotting. Cell lysates were also analyzed by SDS–PAGE and immunoblotting.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Infection, Expressing, shRNA, Selection, Blue Native PAGE, Western Blot, SDS Page

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Mutagenesis, In Vivo, Plasmid Preparation, Software

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1 is a novel ATG5 interactor. A, HEK293T cells were cotransfected with plasmids encoding FLAG-tagged ATG5 and/or non-tagged full-length RACK1 proteins. 48 h after transfection, IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs, and immunoprecipitations were performed using FLAG beads. C, endogenous ATG5 protein was immunoprecipitated from wild-type MEF cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, endogenous RACK1 protein was immunoprecipitated from wild-type MEF cell extracts using anti-RACK1 antibodies that were coupled to protein G Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control mouse serum. E, GST pulldown assay. Glutathione-Sepharose beads that were bound to GST-ATG5 recombinant protein or not were incubated with His-RACK1 recombinant protein and washed. Input, immunoblotting of recombinant proteins; GST pulldown, proteins after pulldown. Note that His-RACK1 did not bind to beads alone. F, HEK293T cells were cultured on coverslides and cotransfected with GFP-tagged RACK1 (green) and Cherry-tagged ATG5 (red) constructs. 48 h post-transfection, cells were fixed and analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization. G, non-transfected HEK293T cells were cultured on coverslides. After 72 h of incubation, cells were fixed, and endogenous RACK1 and ATG5 proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) or anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Transfection, Western Blot, Construct, Immunoprecipitation, GST Pulldown Assay, Recombinant, Incubation, Cell Culture, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1 is a novel component of a large ATG12-5-16 protein complex. A, non-transfected HEK293T cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column. Chromatography fractions (F1–13) were separated in SDS-polyacrylamide gels and immunoblotted using anti-ATG16, anti-ATG5, and anti-RACK1 antibodies. CNT, DMSO carrier control; TORIN, torin 1 treatment (250 nm, 3 h); ATG16, ATG16L1; L, total cell lysate; F1 and F2, >800-kDa fractions; F3–6, 800–669-kDa fractions; F7–10, 669–443-kDa fractions; F11 and F12, 443–200-kDa fractions; F13, 200–150-kDa fraction. No protein complexes were detected in lower molecular weight fractions. B, N2A cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column as in A. C, chromatogram showing peaks of the molecular weight marker mix (Sigma, catalog no. MWGF1000); Ve, elution volume. D, OD595 absorbance confirmation of the peaks. E, standardization of the gel filtration column by Ve/V0. V0, void volume. F, curve showing correlation of fractions with protein sizes in kDa. G and H, representative chromatograms obtained for HEK293T (G) and N2A (H) cell lines. I, Tri-SILAC-LC-MS/MS analyses. ATG5 enrichment compared with beads alone (upper panel); enrichment of RACK-ATG5 complex under torin-treated conditions compared with DMSO-treated control (lower panel) (mean ± S.D. of independent experiments, n = 3, *, p < 0.05).

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Transfection, Filtration, Column Chromatography, Molecular Weight, Marker, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Dynamic nature of RACK1-ATG5 interaction under autophagy-inducing conditions. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h). IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kDa. β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and starved in EBSS (2 h) or not. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (2 h). Endogenous ATG5 protein was immunoprecipitated from cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, HEK293T cells were cultured on coverslides. They were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) and anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and ATG5 colocalization. E, HEK293T cells were cultured on coverslides. Cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained by using anti-RACK1 and anti-LC3 primary antibodies. Cells were analyzed under a confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and LC3 co-localization.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Construct, Western Blot, Immunoprecipitation, Cell Culture, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1 is required for mTOR inhibition and starvation-induced autophagy, but it is not an autophagy target. A, HEK293T cells were cultured on coverslides and transfected with siRACK1 or control siRNA (CNT siRNA). 48 h post-transfection, cells were treated or not (−) with rapamycin (Rapa, 200 nm, 16 h) or starved in EBSS (2 h) in the presence or absence of BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05; **, p < 0.01). Endogenous protein expression levels were checked in cell extracts from the same experiments using anti-p62, anti-LC3, and anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, representative immunofluorescence pictures of LC3 quantification experiments in A. (−), non-treated cells. White arrows show LC3 dots. C, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the presence of translation inhibitor cycloheximide (0.5 μg/ml). Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control. D, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the absence of translation inhibitor cycloheximide. Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Inhibition, Cell Culture, Transfection, Expressing, Immunofluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Role of mTOR-p70S6K pathway in the regulation of RACK1-ATG5 interaction. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and/or an mTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and/or an shmTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 and/or p70S6K wild-type (WT) constructs. IP were performed using FLAG beads. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. D, HEK293T cells were cotransfected with FLAG-ATG5 and non-tagged RACK1 constructs and/or sip70S6 RNAi. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Determination of RACK1 amino acid residues that are critical for the interaction. A, Clustal Omega alignments of RACK1 protein sequences. Putative p70S6K target RXX(S/T) consensus sequences are highlighted in black boxes. Ser/Thr residue numbers are marked according to Homo sapiens protein sequences. RACK1 GenBankTM reference sequences are as follows: H. sapiens, NP_006089; Mus musculus, NP_032169; Danio rerio, NP_571519; Drosophila melanogaster, AAB72148; Caenorhabditis elegans, NP_501859; Saccharomyces cerevisiae, NP_013834. B, schematic depiction of RACK1 constructs. WD1–7, WD40 domains 1–7. WT RACK1, wild-type RACK1. T39A, S63A, or T128A, mutant RACK1 constructs. Mutated residues were marked. C, HEK293T cells were cotransfected with FLAG-ATG5, non-tagged WT RACK1 or T39A, S63A, T128A RACK1 mutant constructs. IPs were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Construct, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1-ATG5 interaction model. A, schematic representation of human RACK1 (Protein Data Bank code 4AOW). Each WD40 domain (WD1–7) is in a different color. The seven-bladed β-propeller structure is shown. Location of the Ser-63 residue is marked in a square. B, schematic model of RACK1 (Protein Data Bank code 4AOW, silver color) and ATG5 (Protein Data Bank code 4GDK, tan color) interaction. Residues found within 3 Å of the other subunit are selected as binding interface that was rendered in the wire frame surface model (probe radius, 1.4 Å) by coloring ATG5 residues in green and RACK1 in yellow. The region around the Ser-63 residue of RACK1 is shown in a red wire frame and encircled. C, interaction network of predicted RACK1-ATG5 model. D–F, native and mutated RACK1s (S63A and S63D) were energy-minimized and equilibrated in MD simulations. Snapshots of the binding interface are shown with Ser-63, Asp-6, and Lys-38 in licorice models (C, cyan; O, red; N, blue). Structural integrity of binding interface was probed by the distance of the ionic interaction between Asp-6 of ATG5 and Lys-38 of RACK1. Although the wild-type (native) and S63D complexes possessed an intact binding surface with Asp-6–Lys-38 ionic pairing (D and E), S63A showed an extension in Asp-6–Lys-38 distance (F), implying a weakened interaction of RACK1 and ATG5. G, r.m.s.d. of the backbone atoms carbon, nitrogen, and α-carbon. Native, wild-type RACK1; S63A, S63A RACK1; S63D, S63D RACK1. H, fluctuations of ATG5 during 5 ns of MD simulations. S63A RACK1 mutant displayed increased fluctuations at two distinct regions (residues from 32 to 36 and from 50 to 54), which are found at the binding interface (right panel), the observation that suggests that S63A RACK1 destabilizes the ATG5-RACK1 complex.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1-ATG5 interaction is necessary for mTOR inhibition- or starvation-induced autophagy in Neuro2A cells. Cells were cultured on coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. A, 48 h post-transfection, Neuro2A cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. B, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots. C, 48 h post-transfection, Neuro2A cells were cultured in full medium (non-STV) or starved in EBSS (STV, 2 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. D, representative immunofluorescence pictures of LC3 quantification experiments in C. White arrows show LC3 dots.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Inhibition, Cell Culture, Transfection, Plasmid Preparation, Mutagenesis, Construct, Expressing, Western Blot, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Rescue experiments with RACK1 mutant constructs following siRNA knockdown of RACK1. A, HEK293T cells were transfected with siRACK1 or control siRNA (CNT siRNA). The effect of siRNAs was checked by immunoblotting using anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were grown onto coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. 48 h post-transfection, HEK293T cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without E64D (E64D, 10 μg/ml, 1 h) and PepA (pepstatin A, 10 μg/ml, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels were detected by immunoblotting using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. C, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Mutagenesis, Construct, Transfection, Western Blot, Plasmid Preparation, Expressing, Immunofluorescence