Structured Review

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Small Angle X-ray Scattering (SAXS) results . (A) Experimental scattering curve of <t>6xHis-RACK1</t> (open circles) and the theoretical fitting (solid line) by using the program GNOM. Inset : Guinier Region and linear regression (solid line) for R g evaluation. (B) Pair distance distribution function p(r). Inset : Kratky plot.
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1) Product Images from "Solution structure of the human signaling protein RACK1"

Article Title: Solution structure of the human signaling protein RACK1

Journal: BMC Structural Biology

doi: 10.1186/1472-6807-10-15

Small Angle X-ray Scattering (SAXS) results . (A) Experimental scattering curve of 6xHis-RACK1 (open circles) and the theoretical fitting (solid line) by using the program GNOM. Inset : Guinier Region and linear regression (solid line) for R g evaluation. (B) Pair distance distribution function p(r). Inset : Kratky plot.
Figure Legend Snippet: Small Angle X-ray Scattering (SAXS) results . (A) Experimental scattering curve of 6xHis-RACK1 (open circles) and the theoretical fitting (solid line) by using the program GNOM. Inset : Guinier Region and linear regression (solid line) for R g evaluation. (B) Pair distance distribution function p(r). Inset : Kratky plot.

Techniques Used:

Sequence alignment and ab initio model for human 6xHis-RACK1 protein in solution . (A) Alignment between amino acid sequences of RACK1 from Homo sapiens (GI:83641897) versus RACK1A from A. thaliana (GI:30685669). The symbols: * showing identical residues, : residues with high similarity and residues with low similarity. (B) Experimental scattering curve of human 6xHis-RACK1 (open circles) and the calculated scattering intensity from the RACK1 from A. thaliana (solid green line) and Homo sapiens (solid red line) by using the program CRYSOL. The solid blue line represents the scattering intensity calculated from the ab initio model of 6xHis-RACK1. (C) Two orthogonal views of the homology model of human RACK1 (cartoon representation with secondary structure elements) superposed to the three dimensional low resolution ab initio model (semi-transparent yellow surface) of the human RACK1, obtained from processing the SAXS data.
Figure Legend Snippet: Sequence alignment and ab initio model for human 6xHis-RACK1 protein in solution . (A) Alignment between amino acid sequences of RACK1 from Homo sapiens (GI:83641897) versus RACK1A from A. thaliana (GI:30685669). The symbols: * showing identical residues, : residues with high similarity and residues with low similarity. (B) Experimental scattering curve of human 6xHis-RACK1 (open circles) and the calculated scattering intensity from the RACK1 from A. thaliana (solid green line) and Homo sapiens (solid red line) by using the program CRYSOL. The solid blue line represents the scattering intensity calculated from the ab initio model of 6xHis-RACK1. (C) Two orthogonal views of the homology model of human RACK1 (cartoon representation with secondary structure elements) superposed to the three dimensional low resolution ab initio model (semi-transparent yellow surface) of the human RACK1, obtained from processing the SAXS data.

Techniques Used: Sequencing

RACK1 sedimentation velocity experiments . (A) Sedimentation velocity experiments were carried out at 4°C, 40,000 rpm (AN-60Ti rotor), and with scan data acquisition at 272 nm. Data were fitted using SedFit software (Version 11.8). Figure displays the c(S) fitting for RACK1 at concentrations of 0.1, 0.2 and 0.3 mg/mL. The c(S) curves suggest that RACK1 present a predominant specie and three or four others species with higher sedimentation coefficients ( inset ) (B) Plots of s 20,ω (of the predominant specie) versus protein concentration fitted by linear regression to calculate the s 0 20;w: 3.00 ± 0.01 Svedberg.
Figure Legend Snippet: RACK1 sedimentation velocity experiments . (A) Sedimentation velocity experiments were carried out at 4°C, 40,000 rpm (AN-60Ti rotor), and with scan data acquisition at 272 nm. Data were fitted using SedFit software (Version 11.8). Figure displays the c(S) fitting for RACK1 at concentrations of 0.1, 0.2 and 0.3 mg/mL. The c(S) curves suggest that RACK1 present a predominant specie and three or four others species with higher sedimentation coefficients ( inset ) (B) Plots of s 20,ω (of the predominant specie) versus protein concentration fitted by linear regression to calculate the s 0 20;w: 3.00 ± 0.01 Svedberg.

Techniques Used: Sedimentation, Software, Protein Concentration

 RACK1  hydrodynamic properties.
Figure Legend Snippet: RACK1 hydrodynamic properties.

Techniques Used:

RACK1 Sedimentation equilibrium experiments . (A) The figure shows the best fits of experimental data for 300 μg/mL of RACK1 (4 °C) at 12000, 15000, and 18000 rpm with the self-association methods (SedPhat program - Material and Methods). The random distribution of the residuals (bottom panel) indicates that the fit is satisfactory. Sedimentation equilibrium data of RACK1 agree with a monomer structure with 37.2 kDa in equilibrium with of dimers and tetramers (see Results and Discussion for details). (B) Sedimentation equilibrium interaction between RACK1 and KI-1/57(122-413) was tested at 300 μg/mL of both proteins at 12,000, 15,000, and 18,000 rpm and 4 °C. The data were fitted by self-association method (SedPhat program - Material and Methods) and resulted in random distribution of the residuals (bottom panel) which indicates that the fit is satisfactory. The fitting data suggest that RACK1 and Ki-1/57(122-413) interacts with an affinity constant of (1.5 ± 0.2) × 10 6 M -1 .
Figure Legend Snippet: RACK1 Sedimentation equilibrium experiments . (A) The figure shows the best fits of experimental data for 300 μg/mL of RACK1 (4 °C) at 12000, 15000, and 18000 rpm with the self-association methods (SedPhat program - Material and Methods). The random distribution of the residuals (bottom panel) indicates that the fit is satisfactory. Sedimentation equilibrium data of RACK1 agree with a monomer structure with 37.2 kDa in equilibrium with of dimers and tetramers (see Results and Discussion for details). (B) Sedimentation equilibrium interaction between RACK1 and KI-1/57(122-413) was tested at 300 μg/mL of both proteins at 12,000, 15,000, and 18,000 rpm and 4 °C. The data were fitted by self-association method (SedPhat program - Material and Methods) and resulted in random distribution of the residuals (bottom panel) which indicates that the fit is satisfactory. The fitting data suggest that RACK1 and Ki-1/57(122-413) interacts with an affinity constant of (1.5 ± 0.2) × 10 6 M -1 .

Techniques Used: Sedimentation

Titration of Ki-1/57(122-413) into RACK1 solution monitored by fluorescence . (A) Fluorescence emission spectra titration of purified 6xHis RACK1 protein (1 μM) with titration of Ki-1/57 at 0.1 μM (black, filled square), 0.2 μM (black, filled circle), 0.3 μM (black, filled triangle), 0.5 μM (open, white triangle) and 1 μM (open, white circle). The excitation wavelength was 295 nm and spectra were background corrected. Inset : Area under curve (AUC) of RACK1 fluorescence emission spectrum in function of Ki-1/57(122-413) concentration. The arrow indicates the increase on the RACK1 fluorescence signal at Ki-1/57(122-413) 0.3 uM. ( B ) The titration curve was represented as difference of fluorescence intensity at 334 nm θ (black, filled square) as function of Ki-1/57(122-413) concentration in the range from 0.3 μM and 1.4 μM and fitted with the modified Hill equation (Equation 5). The dashed line represents the fitting curve. The KD between RACK1 and Ki-1/57(122-413) was of around 0.60 μM which corresponds to the dissociation constant of RACK1\Ki-1/57(122-413) observed in sedimentation equilibrium experiments. The number of cooperative sites n was 8 ± 1.
Figure Legend Snippet: Titration of Ki-1/57(122-413) into RACK1 solution monitored by fluorescence . (A) Fluorescence emission spectra titration of purified 6xHis RACK1 protein (1 μM) with titration of Ki-1/57 at 0.1 μM (black, filled square), 0.2 μM (black, filled circle), 0.3 μM (black, filled triangle), 0.5 μM (open, white triangle) and 1 μM (open, white circle). The excitation wavelength was 295 nm and spectra were background corrected. Inset : Area under curve (AUC) of RACK1 fluorescence emission spectrum in function of Ki-1/57(122-413) concentration. The arrow indicates the increase on the RACK1 fluorescence signal at Ki-1/57(122-413) 0.3 uM. ( B ) The titration curve was represented as difference of fluorescence intensity at 334 nm θ (black, filled square) as function of Ki-1/57(122-413) concentration in the range from 0.3 μM and 1.4 μM and fitted with the modified Hill equation (Equation 5). The dashed line represents the fitting curve. The KD between RACK1 and Ki-1/57(122-413) was of around 0.60 μM which corresponds to the dissociation constant of RACK1\Ki-1/57(122-413) observed in sedimentation equilibrium experiments. The number of cooperative sites n was 8 ± 1.

Techniques Used: Titration, Fluorescence, Purification, Concentration Assay, Modification, Sedimentation


Structured Review

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<t>RACK1</t> is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.
Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
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1) Product Images from "Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling"

Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

Journal: International Journal of Oncology

doi: 10.3892/ijo.2022.5373

RACK1 is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.
Figure Legend Snippet: RACK1 is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.

Techniques Used: Migration, In Vitro, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Real-time Polymerase Chain Reaction

RACK1 enhances the glycolysis in cervical cancer cells. (A) Common differential metabolites in the metabolic pathway of the supernatant of shRACK1/MS751 and shRACK1/SiHa cells. Aminoacyl-tRNA biosynthesis; Valine, leucine and isoleucine biosynthesis; glycolysis/gluconeogenesis; Glyoxylate and dicarboxylate metabolism; glycine, serine and threonine metabolism; arginine and proline metabolism; valine, leucine and isoleucine degradation; butanoate metabolism; pyruvate metabolism; glutathione metabolism. (B) Effect of RACK1 on glucose uptake and lactate production. (C) Effect of RACK1 on the expression of the AKT/mTOR pathway, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using western blot analysis (left panel), with the protein bands assessed (right panel). (D) Effect of RACK1 on the mRNA expression of the GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using reverse transcription-quantitative PCR analysis. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shNON group ( * P<0.05, ** P<0.01 and *** P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2.
Figure Legend Snippet: RACK1 enhances the glycolysis in cervical cancer cells. (A) Common differential metabolites in the metabolic pathway of the supernatant of shRACK1/MS751 and shRACK1/SiHa cells. Aminoacyl-tRNA biosynthesis; Valine, leucine and isoleucine biosynthesis; glycolysis/gluconeogenesis; Glyoxylate and dicarboxylate metabolism; glycine, serine and threonine metabolism; arginine and proline metabolism; valine, leucine and isoleucine degradation; butanoate metabolism; pyruvate metabolism; glutathione metabolism. (B) Effect of RACK1 on glucose uptake and lactate production. (C) Effect of RACK1 on the expression of the AKT/mTOR pathway, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using western blot analysis (left panel), with the protein bands assessed (right panel). (D) Effect of RACK1 on the mRNA expression of the GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using reverse transcription-quantitative PCR analysis. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shNON group ( * P<0.05, ** P<0.01 and *** P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2.

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

RACK1 interacts with IGF1R promotes the glycolysis, aggressiveness and lymphangiogenesis by activating IGF1R/AKT/mTOR signaling in MS751 cells. (A) Venn diagram showing the factors interacting with RACK1, co-existing in the Hitpredict and Genemania databases. (B) Association between RACK1 and IGF1R expression was analyzed using immunofluorescence staining in MS751 and SiHa cells. Nuclei were stained with DAPI (blue). (C) Co-IP assays were used to detect the interaction between RACK1 and IGF1R in MS751 cells (upper panel). Co-IP assay was used to detect the interaction between RACK1 and IGF1R in shRACK1/MS751 and shNON/MS751 cells (lower panel). (D) shRACK1/MS751 cells were stimulated for different time periods (6, 12 and 24 h) with 200 ng/ml IGF1. The RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2 and LDHA, E-cadherin and N-cadherin expression was examined using western blot analysis. (E) shRACK1/MS751 cells were stimulated for 24 h with 200 ng/ml IGF1 and various concentrations (10 and 20 nM) of Rapa. The expression of RACK1, p-mTOR (ser2448), mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin was examined using western blot analysis. (F) Transwell assays were performed to investigate the effects of IGF1 and IGF1 combined with Rapa on the invasion and migration of MS751 and HLECs cells (upper panel), with the quantified bands assessed (lower panel). (G) Effect of IGF1 and IGF1 combined with Rapa on tube formation by HLECs. (H) Effect of IGF1 and IGF1 combined with Rapa on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( * P<0.05 and *** P<0.001). or shRACK1 + IGF1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2; Rapa, rapamycin; IGF1, insulin-like growth factor 1; HLECs, human lymphatic endothelial cells.
Figure Legend Snippet: RACK1 interacts with IGF1R promotes the glycolysis, aggressiveness and lymphangiogenesis by activating IGF1R/AKT/mTOR signaling in MS751 cells. (A) Venn diagram showing the factors interacting with RACK1, co-existing in the Hitpredict and Genemania databases. (B) Association between RACK1 and IGF1R expression was analyzed using immunofluorescence staining in MS751 and SiHa cells. Nuclei were stained with DAPI (blue). (C) Co-IP assays were used to detect the interaction between RACK1 and IGF1R in MS751 cells (upper panel). Co-IP assay was used to detect the interaction between RACK1 and IGF1R in shRACK1/MS751 and shNON/MS751 cells (lower panel). (D) shRACK1/MS751 cells were stimulated for different time periods (6, 12 and 24 h) with 200 ng/ml IGF1. The RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2 and LDHA, E-cadherin and N-cadherin expression was examined using western blot analysis. (E) shRACK1/MS751 cells were stimulated for 24 h with 200 ng/ml IGF1 and various concentrations (10 and 20 nM) of Rapa. The expression of RACK1, p-mTOR (ser2448), mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin was examined using western blot analysis. (F) Transwell assays were performed to investigate the effects of IGF1 and IGF1 combined with Rapa on the invasion and migration of MS751 and HLECs cells (upper panel), with the quantified bands assessed (lower panel). (G) Effect of IGF1 and IGF1 combined with Rapa on tube formation by HLECs. (H) Effect of IGF1 and IGF1 combined with Rapa on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( * P<0.05 and *** P<0.001). or shRACK1 + IGF1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2; Rapa, rapamycin; IGF1, insulin-like growth factor 1; HLECs, human lymphatic endothelial cells.

Techniques Used: Expressing, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Migration

RACK1 is a direct target of POU2F2. (A) Venn diagram showing the transcription factor binding to RACK1 co-existing in UCSC, HUMANTFDB and PROMO database. (B) POU2F2 DNA binding sites are present in the human RACK1 promoter region. The top panel shows the WT and MUT forms of the putative POU2F2 target sequences in RACK1 3′-UTR. Red font (upper panel) refers to the putative POU2F2 targeting sequence in the RACK1 3′-UTR. Red font (lower panel) refers to mutations in the POU2F2 targeting sequence in RACK1 3′-UTR. (C) Luciferase reporter assays of WT and MUT RACK1 luciferase reporters transfected with POU2F2 in MS751 cells. (D) ChIP-PCR assay was used to detect POU2F2-binding sites in the sequence of the RACK1 promoter. (E) Effect of POU2F2 on the expression of RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin in shRACK1/MS751 cells, as evaluated using western blot analysis. (F) POU2F2 OE-shRACK1/MS751 cells were stimulated for 24 h with 10 mM 2-DG. Effect of POU2F2 and 2-DG combined with POU2F2 on the invasion and migration of shRACK1/MS751 cells and migration of HLECs (upper panel), with the quantified bands assessed (lower panel). (G) Effect of POU2F2 on tube formation of HLECs. (H) Effect of POU2F2 on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( ** P<0.01 and *** P<0.001) or OE POU2F2/shRACK1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; POU2F2, POU class 2 homeobox 2; WT, wild-type; MUT, mutant type; 2-DG, 2-deoxy-D-glucose; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; HLECs, human lymphatic endothelial cells; OE, overexpression.
Figure Legend Snippet: RACK1 is a direct target of POU2F2. (A) Venn diagram showing the transcription factor binding to RACK1 co-existing in UCSC, HUMANTFDB and PROMO database. (B) POU2F2 DNA binding sites are present in the human RACK1 promoter region. The top panel shows the WT and MUT forms of the putative POU2F2 target sequences in RACK1 3′-UTR. Red font (upper panel) refers to the putative POU2F2 targeting sequence in the RACK1 3′-UTR. Red font (lower panel) refers to mutations in the POU2F2 targeting sequence in RACK1 3′-UTR. (C) Luciferase reporter assays of WT and MUT RACK1 luciferase reporters transfected with POU2F2 in MS751 cells. (D) ChIP-PCR assay was used to detect POU2F2-binding sites in the sequence of the RACK1 promoter. (E) Effect of POU2F2 on the expression of RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin in shRACK1/MS751 cells, as evaluated using western blot analysis. (F) POU2F2 OE-shRACK1/MS751 cells were stimulated for 24 h with 10 mM 2-DG. Effect of POU2F2 and 2-DG combined with POU2F2 on the invasion and migration of shRACK1/MS751 cells and migration of HLECs (upper panel), with the quantified bands assessed (lower panel). (G) Effect of POU2F2 on tube formation of HLECs. (H) Effect of POU2F2 on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( ** P<0.01 and *** P<0.001) or OE POU2F2/shRACK1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; POU2F2, POU class 2 homeobox 2; WT, wild-type; MUT, mutant type; 2-DG, 2-deoxy-D-glucose; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; HLECs, human lymphatic endothelial cells; OE, overexpression.

Techniques Used: Binding Assay, Sequencing, Luciferase, Transfection, Expressing, Western Blot, Migration, Mutagenesis, Over Expression

RACK1 enhances the LNM of MS751 cells in vivo . (A) Representative images of inguinal lymph nodes in different groups of nude mice (n=8, left panel). Representative images of inguinal lymph nodes of H&E staining in different groups of nude mice (middle panel). Representative images of anti-GFP IHC analysis for inguinal lymph nodes in different groups of nude mice (right panel). (B) Representative images of footpads primary tumor in different groups of nude mice (left panel). Representative images of footpads primary tumor tissues of H&E staining (middle panel) and percentages of PDPN-indicated lymphatic vessels density in different groups of nude mice (right panel). (C) The image of all tumors with a ruler. (D) Representative images of RACK1, GLUT1, PKM2, HK2, LDHA, E-cadherin and N-cadherin expression in footpad primary tumor tissues in IHC analysis. (E) The bar graph summarizes the tumor size assessed (left panel), and lymph node volume assessed (right panel). Data were compared with the shNON group ( ** P<0.01 and *** P<0.001). Data are presented as the mean ± SD and were analyzed using the t-test. IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; PDPN, podoplanin.
Figure Legend Snippet: RACK1 enhances the LNM of MS751 cells in vivo . (A) Representative images of inguinal lymph nodes in different groups of nude mice (n=8, left panel). Representative images of inguinal lymph nodes of H&E staining in different groups of nude mice (middle panel). Representative images of anti-GFP IHC analysis for inguinal lymph nodes in different groups of nude mice (right panel). (B) Representative images of footpads primary tumor in different groups of nude mice (left panel). Representative images of footpads primary tumor tissues of H&E staining (middle panel) and percentages of PDPN-indicated lymphatic vessels density in different groups of nude mice (right panel). (C) The image of all tumors with a ruler. (D) Representative images of RACK1, GLUT1, PKM2, HK2, LDHA, E-cadherin and N-cadherin expression in footpad primary tumor tissues in IHC analysis. (E) The bar graph summarizes the tumor size assessed (left panel), and lymph node volume assessed (right panel). Data were compared with the shNON group ( ** P<0.01 and *** P<0.001). Data are presented as the mean ± SD and were analyzed using the t-test. IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; PDPN, podoplanin.

Techniques Used: In Vivo, Staining, Expressing, Immunohistochemistry

 RACK1  expression in cervical carcinoma according to the histopathological characteristics of the patients with cervical cancer.
Figure Legend Snippet: RACK1 expression in cervical carcinoma according to the histopathological characteristics of the patients with cervical cancer.

Techniques Used: Expressing

Associations between RACK1, IGF1R, POU2F2, and HK2 expression in tissues from patients with CC. Representative images of IHC staining in case 1 for RACK1, IGF1R, POU2F2 and HK2 (strong RACK1 expression). Representative images of IHC staining in case 2 for RACK1, IGF1R, POU2F2 and HK2 (medium RACK1 expression). Representative images of IHC staining in case 3 for RACK1, IGF1R, POU2F2 and HK2 (weak RACK1 expression). Representative images of IHC staining in case 4 for RACK1, IGF1R, POU2F2 and HK2 (negative RACK1 expression). CC, cervical cancer; IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2.
Figure Legend Snippet: Associations between RACK1, IGF1R, POU2F2, and HK2 expression in tissues from patients with CC. Representative images of IHC staining in case 1 for RACK1, IGF1R, POU2F2 and HK2 (strong RACK1 expression). Representative images of IHC staining in case 2 for RACK1, IGF1R, POU2F2 and HK2 (medium RACK1 expression). Representative images of IHC staining in case 3 for RACK1, IGF1R, POU2F2 and HK2 (weak RACK1 expression). Representative images of IHC staining in case 4 for RACK1, IGF1R, POU2F2 and HK2 (negative RACK1 expression). CC, cervical cancer; IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2.

Techniques Used: Expressing, Immunohistochemistry

Schematic illustration of the mechanisms by which the POU2F2/RACK1/IGF1R/AKT/mTOR pathway promotes cell lymph node metastasis dependent on glycolysis. RACK1, receptor for activated C kinase 1; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2.
Figure Legend Snippet: Schematic illustration of the mechanisms by which the POU2F2/RACK1/IGF1R/AKT/mTOR pathway promotes cell lymph node metastasis dependent on glycolysis. RACK1, receptor for activated C kinase 1; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2.

Techniques Used:


Structured Review

Millipore rack1
Model of AeaeRACK1. (A) The predicted protein sequence of AeaeRACK1 was used to construct a three-dimensional model using the human <t>RACK1</t> structure as template (PDB ID: 2zkqa). Each blade of the propeller is made up of four antiparallel β-strands and forms a WD domain, indicated by Roman numerals I to VII. (B) Sequence alignment of the seven WD domains of AeaeRACK1, which are flanked GH or equivalents dipeptides (enclosed in a dashed line box), WD or equivalent dipeptides (enclosed in a continuous line box).
Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rack1/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rack1 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "Aeal RACK1 expression and localization in response to stress in C6/36 HT mosquito cells"

Article Title: Aeal RACK1 expression and localization in response to stress in C6/36 HT mosquito cells

Journal: Journal of proteomics

doi: 10.1016/j.jprot.2014.11.019

Model of AeaeRACK1. (A) The predicted protein sequence of AeaeRACK1 was used to construct a three-dimensional model using the human RACK1 structure as template (PDB ID: 2zkqa). Each blade of the propeller is made up of four antiparallel β-strands and forms a WD domain, indicated by Roman numerals I to VII. (B) Sequence alignment of the seven WD domains of AeaeRACK1, which are flanked GH or equivalents dipeptides (enclosed in a dashed line box), WD or equivalent dipeptides (enclosed in a continuous line box).
Figure Legend Snippet: Model of AeaeRACK1. (A) The predicted protein sequence of AeaeRACK1 was used to construct a three-dimensional model using the human RACK1 structure as template (PDB ID: 2zkqa). Each blade of the propeller is made up of four antiparallel β-strands and forms a WD domain, indicated by Roman numerals I to VII. (B) Sequence alignment of the seven WD domains of AeaeRACK1, which are flanked GH or equivalents dipeptides (enclosed in a dashed line box), WD or equivalent dipeptides (enclosed in a continuous line box).

Techniques Used: Sequencing, Construct

Expression of AealRACK1 in C6/36 HT cells in serum deprivation or dexamethasone treatment. The C6/36 HT cells were serum-deprived (SERUMDEPRIV) or dexamethasone(DEX, 13 μM) treated. (A) Cell lysates were resolved in 1D electrophoresis and Western blot was performed using anti-RACK1 polyclonal antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Lanes: A431-Human carcinoma cell line used as positive control; lane CTL—C6/36 HT cells untreated-control; lane SERUM DEPRIV—cells incubated with serum-free MEM; lane DEX—dexamethasone treatment. Same blot was tested with an anti β-actin antibody as loading control. (B) RACK1 presence was determined by densitometric quantification of the unsaturated images of Western blot. Relative expression was calculated using the corresponding value of β-actin. Experiments were conducted in duplicate (*p = 0.01; **p < 0.05). (C) Total proteins of C6/36 HT cells under serum deprivation treatment were resolved in the first dimension on an IPG strip pH 3 −10 NL and in the second dimension on a 12% SDS-PAGE. Gel was stained with Bio-Safe Coomassie blue. (D) Identification of AealRACK1 by 2-DEWB. A replica 2-DE gel was blotted onto NC membrane and incubated with a specific anti-RACK1 antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Two protein spots (indicated by arrows) recognized by anti-RACK1 were detected. Both proteins spots were identified as RACK1 by MS (Table 1).
Figure Legend Snippet: Expression of AealRACK1 in C6/36 HT cells in serum deprivation or dexamethasone treatment. The C6/36 HT cells were serum-deprived (SERUMDEPRIV) or dexamethasone(DEX, 13 μM) treated. (A) Cell lysates were resolved in 1D electrophoresis and Western blot was performed using anti-RACK1 polyclonal antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Lanes: A431-Human carcinoma cell line used as positive control; lane CTL—C6/36 HT cells untreated-control; lane SERUM DEPRIV—cells incubated with serum-free MEM; lane DEX—dexamethasone treatment. Same blot was tested with an anti β-actin antibody as loading control. (B) RACK1 presence was determined by densitometric quantification of the unsaturated images of Western blot. Relative expression was calculated using the corresponding value of β-actin. Experiments were conducted in duplicate (*p = 0.01; **p < 0.05). (C) Total proteins of C6/36 HT cells under serum deprivation treatment were resolved in the first dimension on an IPG strip pH 3 −10 NL and in the second dimension on a 12% SDS-PAGE. Gel was stained with Bio-Safe Coomassie blue. (D) Identification of AealRACK1 by 2-DEWB. A replica 2-DE gel was blotted onto NC membrane and incubated with a specific anti-RACK1 antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Two protein spots (indicated by arrows) recognized by anti-RACK1 were detected. Both proteins spots were identified as RACK1 by MS (Table 1).

Techniques Used: Expressing, Electrophoresis, Western Blot, Positive Control, Incubation, Stripping Membranes, SDS Page, Staining

Identification of Aeal  RACK1  protein from C6/36 HT cell line by 2-DE/WB and LC-MS/MS.
Figure Legend Snippet: Identification of Aeal RACK1 protein from C6/36 HT cell line by 2-DE/WB and LC-MS/MS.

Techniques Used:

Proteins associated with AealRACK1 during serum deprivation or dexamethasone treatment. Protein extracts of C6/36 HT cells were immunoprecipitated with an anti-RACK1 antibody. (A) Coomassie blue stained proteins resolved by SDS-PAGE. Input: total extracts; IP:IgG, reaction with a non-related antibody. IP: RACK1, immunoprecipitation of cells lysates using an anti-RACK1 antibody. Lanes 1 and 6—C6/36 control cells extract; lanes 2, 4 and 7—serum deprivation; lanes 3, 5 and 8—cells treated with dexamethasone (13 μM). Major protein bands were excised from the gel and identified by MS (arrows 1–7). The presence of RACK1 in total extracts (INPUT) and in immunoprecipitates (IP: RACK1) was verified by immunoblot (IB). (B) Input and IP: RACK1 in the absence (−) or presence (+) of dexamethasone. Panel C: Input and IP:RACK1 in control (−) and serum-deprived cells (+). Lanes IgG—immunoprecipitation negative control conducted with a non-related IgG antibody. MCF7—total extracts of human mammary cancer cells used as positive control.
Figure Legend Snippet: Proteins associated with AealRACK1 during serum deprivation or dexamethasone treatment. Protein extracts of C6/36 HT cells were immunoprecipitated with an anti-RACK1 antibody. (A) Coomassie blue stained proteins resolved by SDS-PAGE. Input: total extracts; IP:IgG, reaction with a non-related antibody. IP: RACK1, immunoprecipitation of cells lysates using an anti-RACK1 antibody. Lanes 1 and 6—C6/36 control cells extract; lanes 2, 4 and 7—serum deprivation; lanes 3, 5 and 8—cells treated with dexamethasone (13 μM). Major protein bands were excised from the gel and identified by MS (arrows 1–7). The presence of RACK1 in total extracts (INPUT) and in immunoprecipitates (IP: RACK1) was verified by immunoblot (IB). (B) Input and IP: RACK1 in the absence (−) or presence (+) of dexamethasone. Panel C: Input and IP:RACK1 in control (−) and serum-deprived cells (+). Lanes IgG—immunoprecipitation negative control conducted with a non-related IgG antibody. MCF7—total extracts of human mammary cancer cells used as positive control.

Techniques Used: Immunoprecipitation, Staining, SDS Page, Western Blot, Negative Control, Positive Control

Proteins associated to Aeae  RACK1  in serum-deprived C6/36 HT cells identified by LC/MS/MS.
Figure Legend Snippet: Proteins associated to Aeae RACK1 in serum-deprived C6/36 HT cells identified by LC/MS/MS.

Techniques Used: Sequencing, Binding Assay, Activity Assay, Transduction, De-Phosphorylation Assay


Figure Legend Snippet: Functional classification of all 33 identified proteins by immunoprecipitation and MS.

Techniques Used: Functional Assay, Immunoprecipitation, DNA Synthesis, Binding Assay

Localization of AealRACK1 in C6/36 cells under serum deprivation and dexamethasone treated. C6/36 HT cells untreated (A–C), serum-deprived (D–F) or treated with dexamethasone (13 μM) (G–I), were fixed with 1% paraformaldehyde and permeabilized with ice-cold acetone. The AealRACK1 protein was localized using anti-RACK1 polyclonal antibody and with Alexa Fluor 488-conjugated secondary antibody (green color) (A, D and G). Nuclei were DAPI stained (blue color) (B, E and H). Merge images (C, F and I). Cells were examined using confocal microscopy.
Figure Legend Snippet: Localization of AealRACK1 in C6/36 cells under serum deprivation and dexamethasone treated. C6/36 HT cells untreated (A–C), serum-deprived (D–F) or treated with dexamethasone (13 μM) (G–I), were fixed with 1% paraformaldehyde and permeabilized with ice-cold acetone. The AealRACK1 protein was localized using anti-RACK1 polyclonal antibody and with Alexa Fluor 488-conjugated secondary antibody (green color) (A, D and G). Nuclei were DAPI stained (blue color) (B, E and H). Merge images (C, F and I). Cells were examined using confocal microscopy.

Techniques Used: Staining, Confocal Microscopy


Structured Review

Millipore rack1
Model of AeaeRACK1. (A) The predicted protein sequence of AeaeRACK1 was used to construct a three-dimensional model using the human <t>RACK1</t> structure as template (PDB ID: 2zkqa). Each blade of the propeller is made up of four antiparallel β-strands and forms a WD domain, indicated by Roman numerals I to VII. (B) Sequence alignment of the seven WD domains of AeaeRACK1, which are flanked GH or equivalents dipeptides (enclosed in a dashed line box), WD or equivalent dipeptides (enclosed in a continuous line box).
Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rack1/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rack1 - by Bioz Stars, 2024-04
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Images

1) Product Images from "Aeal RACK1 expression and localization in response to stress in C6/36 HT mosquito cells"

Article Title: Aeal RACK1 expression and localization in response to stress in C6/36 HT mosquito cells

Journal: Journal of proteomics

doi: 10.1016/j.jprot.2014.11.019

Model of AeaeRACK1. (A) The predicted protein sequence of AeaeRACK1 was used to construct a three-dimensional model using the human RACK1 structure as template (PDB ID: 2zkqa). Each blade of the propeller is made up of four antiparallel β-strands and forms a WD domain, indicated by Roman numerals I to VII. (B) Sequence alignment of the seven WD domains of AeaeRACK1, which are flanked GH or equivalents dipeptides (enclosed in a dashed line box), WD or equivalent dipeptides (enclosed in a continuous line box).
Figure Legend Snippet: Model of AeaeRACK1. (A) The predicted protein sequence of AeaeRACK1 was used to construct a three-dimensional model using the human RACK1 structure as template (PDB ID: 2zkqa). Each blade of the propeller is made up of four antiparallel β-strands and forms a WD domain, indicated by Roman numerals I to VII. (B) Sequence alignment of the seven WD domains of AeaeRACK1, which are flanked GH or equivalents dipeptides (enclosed in a dashed line box), WD or equivalent dipeptides (enclosed in a continuous line box).

Techniques Used: Sequencing, Construct

Expression of AealRACK1 in C6/36 HT cells in serum deprivation or dexamethasone treatment. The C6/36 HT cells were serum-deprived (SERUMDEPRIV) or dexamethasone(DEX, 13 μM) treated. (A) Cell lysates were resolved in 1D electrophoresis and Western blot was performed using anti-RACK1 polyclonal antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Lanes: A431-Human carcinoma cell line used as positive control; lane CTL—C6/36 HT cells untreated-control; lane SERUM DEPRIV—cells incubated with serum-free MEM; lane DEX—dexamethasone treatment. Same blot was tested with an anti β-actin antibody as loading control. (B) RACK1 presence was determined by densitometric quantification of the unsaturated images of Western blot. Relative expression was calculated using the corresponding value of β-actin. Experiments were conducted in duplicate (*p = 0.01; **p < 0.05). (C) Total proteins of C6/36 HT cells under serum deprivation treatment were resolved in the first dimension on an IPG strip pH 3 −10 NL and in the second dimension on a 12% SDS-PAGE. Gel was stained with Bio-Safe Coomassie blue. (D) Identification of AealRACK1 by 2-DEWB. A replica 2-DE gel was blotted onto NC membrane and incubated with a specific anti-RACK1 antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Two protein spots (indicated by arrows) recognized by anti-RACK1 were detected. Both proteins spots were identified as RACK1 by MS (Table 1).
Figure Legend Snippet: Expression of AealRACK1 in C6/36 HT cells in serum deprivation or dexamethasone treatment. The C6/36 HT cells were serum-deprived (SERUMDEPRIV) or dexamethasone(DEX, 13 μM) treated. (A) Cell lysates were resolved in 1D electrophoresis and Western blot was performed using anti-RACK1 polyclonal antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Lanes: A431-Human carcinoma cell line used as positive control; lane CTL—C6/36 HT cells untreated-control; lane SERUM DEPRIV—cells incubated with serum-free MEM; lane DEX—dexamethasone treatment. Same blot was tested with an anti β-actin antibody as loading control. (B) RACK1 presence was determined by densitometric quantification of the unsaturated images of Western blot. Relative expression was calculated using the corresponding value of β-actin. Experiments were conducted in duplicate (*p = 0.01; **p < 0.05). (C) Total proteins of C6/36 HT cells under serum deprivation treatment were resolved in the first dimension on an IPG strip pH 3 −10 NL and in the second dimension on a 12% SDS-PAGE. Gel was stained with Bio-Safe Coomassie blue. (D) Identification of AealRACK1 by 2-DEWB. A replica 2-DE gel was blotted onto NC membrane and incubated with a specific anti-RACK1 antibody followed by an anti-IgG-rabbit polyclonal antibody coupled to peroxidase. Two protein spots (indicated by arrows) recognized by anti-RACK1 were detected. Both proteins spots were identified as RACK1 by MS (Table 1).

Techniques Used: Expressing, Electrophoresis, Western Blot, Positive Control, Incubation, Stripping Membranes, SDS Page, Staining

Identification of Aeal  RACK1  protein from C6/36 HT cell line by 2-DE/WB and LC-MS/MS.
Figure Legend Snippet: Identification of Aeal RACK1 protein from C6/36 HT cell line by 2-DE/WB and LC-MS/MS.

Techniques Used:

Proteins associated with AealRACK1 during serum deprivation or dexamethasone treatment. Protein extracts of C6/36 HT cells were immunoprecipitated with an anti-RACK1 antibody. (A) Coomassie blue stained proteins resolved by SDS-PAGE. Input: total extracts; IP:IgG, reaction with a non-related antibody. IP: RACK1, immunoprecipitation of cells lysates using an anti-RACK1 antibody. Lanes 1 and 6—C6/36 control cells extract; lanes 2, 4 and 7—serum deprivation; lanes 3, 5 and 8—cells treated with dexamethasone (13 μM). Major protein bands were excised from the gel and identified by MS (arrows 1–7). The presence of RACK1 in total extracts (INPUT) and in immunoprecipitates (IP: RACK1) was verified by immunoblot (IB). (B) Input and IP: RACK1 in the absence (−) or presence (+) of dexamethasone. Panel C: Input and IP:RACK1 in control (−) and serum-deprived cells (+). Lanes IgG—immunoprecipitation negative control conducted with a non-related IgG antibody. MCF7—total extracts of human mammary cancer cells used as positive control.
Figure Legend Snippet: Proteins associated with AealRACK1 during serum deprivation or dexamethasone treatment. Protein extracts of C6/36 HT cells were immunoprecipitated with an anti-RACK1 antibody. (A) Coomassie blue stained proteins resolved by SDS-PAGE. Input: total extracts; IP:IgG, reaction with a non-related antibody. IP: RACK1, immunoprecipitation of cells lysates using an anti-RACK1 antibody. Lanes 1 and 6—C6/36 control cells extract; lanes 2, 4 and 7—serum deprivation; lanes 3, 5 and 8—cells treated with dexamethasone (13 μM). Major protein bands were excised from the gel and identified by MS (arrows 1–7). The presence of RACK1 in total extracts (INPUT) and in immunoprecipitates (IP: RACK1) was verified by immunoblot (IB). (B) Input and IP: RACK1 in the absence (−) or presence (+) of dexamethasone. Panel C: Input and IP:RACK1 in control (−) and serum-deprived cells (+). Lanes IgG—immunoprecipitation negative control conducted with a non-related IgG antibody. MCF7—total extracts of human mammary cancer cells used as positive control.

Techniques Used: Immunoprecipitation, Staining, SDS Page, Western Blot, Negative Control, Positive Control

Proteins associated to Aeae  RACK1  in serum-deprived C6/36 HT cells identified by LC/MS/MS.
Figure Legend Snippet: Proteins associated to Aeae RACK1 in serum-deprived C6/36 HT cells identified by LC/MS/MS.

Techniques Used: Sequencing, Binding Assay, Activity Assay, Transduction, De-Phosphorylation Assay


Figure Legend Snippet: Functional classification of all 33 identified proteins by immunoprecipitation and MS.

Techniques Used: Functional Assay, Immunoprecipitation, DNA Synthesis, Binding Assay

Localization of AealRACK1 in C6/36 cells under serum deprivation and dexamethasone treated. C6/36 HT cells untreated (A–C), serum-deprived (D–F) or treated with dexamethasone (13 μM) (G–I), were fixed with 1% paraformaldehyde and permeabilized with ice-cold acetone. The AealRACK1 protein was localized using anti-RACK1 polyclonal antibody and with Alexa Fluor 488-conjugated secondary antibody (green color) (A, D and G). Nuclei were DAPI stained (blue color) (B, E and H). Merge images (C, F and I). Cells were examined using confocal microscopy.
Figure Legend Snippet: Localization of AealRACK1 in C6/36 cells under serum deprivation and dexamethasone treated. C6/36 HT cells untreated (A–C), serum-deprived (D–F) or treated with dexamethasone (13 μM) (G–I), were fixed with 1% paraformaldehyde and permeabilized with ice-cold acetone. The AealRACK1 protein was localized using anti-RACK1 polyclonal antibody and with Alexa Fluor 488-conjugated secondary antibody (green color) (A, D and G). Nuclei were DAPI stained (blue color) (B, E and H). Merge images (C, F and I). Cells were examined using confocal microscopy.

Techniques Used: Staining, Confocal Microscopy


Structured Review

Millipore rack1
Disruption of <t>RACK1,</t> AKAP and MAP2 complexes after in vivo intra-RSC infusions of Tat peptides. ( a ) NRB/RACK1 interaction after Tat-RACK1 infusions. ( b ) PKA/AKAP5 interaction after Tat-AKAP infusions. ( c ) PKA/MAP2 interaction after Tat-MAP2 infusions. ** P <0.01 vs scrambled peptide. AKAP, A-kinase anchor protein; MAP, microtubule-associated protein; PKA, protein kinase; RACK1, receptor for activated C kinase 1; RSC, retrosplenial cortex.
Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rack1/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rack1 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling"

Article Title: Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling

Journal: Translational Psychiatry

doi: 10.1038/tp.2015.150

Disruption of RACK1, AKAP and MAP2 complexes after in vivo intra-RSC infusions of Tat peptides. ( a ) NRB/RACK1 interaction after Tat-RACK1 infusions. ( b ) PKA/AKAP5 interaction after Tat-AKAP infusions. ( c ) PKA/MAP2 interaction after Tat-MAP2 infusions. ** P <0.01 vs scrambled peptide. AKAP, A-kinase anchor protein; MAP, microtubule-associated protein; PKA, protein kinase; RACK1, receptor for activated C kinase 1; RSC, retrosplenial cortex.
Figure Legend Snippet: Disruption of RACK1, AKAP and MAP2 complexes after in vivo intra-RSC infusions of Tat peptides. ( a ) NRB/RACK1 interaction after Tat-RACK1 infusions. ( b ) PKA/AKAP5 interaction after Tat-AKAP infusions. ( c ) PKA/MAP2 interaction after Tat-MAP2 infusions. ** P <0.01 vs scrambled peptide. AKAP, A-kinase anchor protein; MAP, microtubule-associated protein; PKA, protein kinase; RACK1, receptor for activated C kinase 1; RSC, retrosplenial cortex.

Techniques Used: In Vivo

Effects of disrupting NR2B/RACK1, AKAP/PKA or MAP2/PKA complexes in RSC on extinction of remotely acquired fear. ( a ) Experimental design and treatment schedule. ( b ) Effects of post-test RSC infusions of Tat-RACK1, Tat-AKAP and Tat-MAP2 on freezing behavior. * P <0.05 vs scrambled peptide. AKAP, A-kinase anchor protein; MAP, microtubule-associated protein; PKA, protein kinase; RACK1, receptor for activated C kinase 1; RSC, retrosplenial cortex.
Figure Legend Snippet: Effects of disrupting NR2B/RACK1, AKAP/PKA or MAP2/PKA complexes in RSC on extinction of remotely acquired fear. ( a ) Experimental design and treatment schedule. ( b ) Effects of post-test RSC infusions of Tat-RACK1, Tat-AKAP and Tat-MAP2 on freezing behavior. * P <0.05 vs scrambled peptide. AKAP, A-kinase anchor protein; MAP, microtubule-associated protein; PKA, protein kinase; RACK1, receptor for activated C kinase 1; RSC, retrosplenial cortex.

Techniques Used:

Effect of disrupting NR2B/RACK1, AKAP/PKA or MAP2/PKA complexes in RSC on depression-related behavior. ( a ) Experimental design and treatment schedule for the novelty-suppressed feeding test. ( b ) Effects of post-test RSC infusions of Tat-RACK1, Tat-AKAP and Tat-MAP2 latency to eat (left) and total distance traveled (right). ( c ) Experimental design for the antidepressant response to MK-801. ( d ) Effects of of Tat-Rack1, Tat-AKAP and Tat-MAP2 on the latency to eat (left) and distance traveled (right) in the novelty-suppressed feeding test following injection of MK-801. * P <0.05, ** P <0.01 vs Scrambled+saline control; # P <0.05 vs Scrambled+MK-801 group. AKAP, A-kinase anchor protein; MAP, microtubule-associated protein; PKA, protein kinase; RACK1, receptor for activated C kinase 1; RSC, retrosplenial cortex.
Figure Legend Snippet: Effect of disrupting NR2B/RACK1, AKAP/PKA or MAP2/PKA complexes in RSC on depression-related behavior. ( a ) Experimental design and treatment schedule for the novelty-suppressed feeding test. ( b ) Effects of post-test RSC infusions of Tat-RACK1, Tat-AKAP and Tat-MAP2 latency to eat (left) and total distance traveled (right). ( c ) Experimental design for the antidepressant response to MK-801. ( d ) Effects of of Tat-Rack1, Tat-AKAP and Tat-MAP2 on the latency to eat (left) and distance traveled (right) in the novelty-suppressed feeding test following injection of MK-801. * P <0.05, ** P <0.01 vs Scrambled+saline control; # P <0.05 vs Scrambled+MK-801 group. AKAP, A-kinase anchor protein; MAP, microtubule-associated protein; PKA, protein kinase; RACK1, receptor for activated C kinase 1; RSC, retrosplenial cortex.

Techniques Used: Injection

Effect of disrupting NR2B/RACK1, AKAP/PKA or MAP2/PKA complexes on the levels of BDNF transcripts. ( a ) Tat peptides administered without (left) or with (right) MK-801 treatment did not affect the expression of Bdnf transcripts. ( b ) Comparisons of normalized levels of the individual transcripts showed in a . * P <0.05 vs Scrambled+saline control. AKAP, A-kinase anchor protein; BDNF, brain-derived neurotrophic factor; MAP, microtubule-associated protein; mRNA, messenger RNA; PKA, protein kinase A; RACK1, receptor for activated C kinase 1.
Figure Legend Snippet: Effect of disrupting NR2B/RACK1, AKAP/PKA or MAP2/PKA complexes on the levels of BDNF transcripts. ( a ) Tat peptides administered without (left) or with (right) MK-801 treatment did not affect the expression of Bdnf transcripts. ( b ) Comparisons of normalized levels of the individual transcripts showed in a . * P <0.05 vs Scrambled+saline control. AKAP, A-kinase anchor protein; BDNF, brain-derived neurotrophic factor; MAP, microtubule-associated protein; mRNA, messenger RNA; PKA, protein kinase A; RACK1, receptor for activated C kinase 1.

Techniques Used: Expressing, Derivative Assay


Structured Review

Millipore anti rack1 rabbit antibody
Anti Rack1 Rabbit Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti rack1 rabbit antibody/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti rack1 rabbit antibody - by Bioz Stars, 2024-04
86/100 stars

Images


Structured Review

Millipore rack1
Table 1.
Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rack1/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rack1 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator"

Article Title: RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00026.2013

Table 1.
Figure Legend Snippet: Table 1.

Techniques Used:


Structured Review

Millipore rack1
Table 1.
Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rack1/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rack1 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator"

Article Title: RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00026.2013

Table 1.
Figure Legend Snippet: Table 1.

Techniques Used:


Structured Review

Millipore rack1
Table 1.
Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rack1/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rack1 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator"

Article Title: RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00026.2013

Table 1.
Figure Legend Snippet: Table 1.

Techniques Used:


Structured Review

Millipore anti rack1
Anti Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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  • 86
    Millipore rack1 full length
    Small Angle X-ray Scattering (SAXS) results . (A) Experimental scattering curve of <t>6xHis-RACK1</t> (open circles) and the theoretical fitting (solid line) by using the program GNOM. Inset : Guinier Region and linear regression (solid line) for R g evaluation. (B) Pair distance distribution function p(r). Inset : Kratky plot.
    Rack1 Full Length, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rack1 full length/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rack1 full length - by Bioz Stars, 2024-04
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    86
    Millipore rack1
    <t>RACK1</t> is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.
    Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rack1/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rack1 - by Bioz Stars, 2024-04
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    86
    Millipore anti rack1 rabbit antibody
    <t>RACK1</t> is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.
    Anti Rack1 Rabbit Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rack1 rabbit antibody/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rack1 rabbit antibody - by Bioz Stars, 2024-04
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    86
    Millipore anti rack1
    <t>RACK1</t> is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.
    Anti Rack1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rack1/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rack1 - by Bioz Stars, 2024-04
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    Image Search Results


    Small Angle X-ray Scattering (SAXS) results . (A) Experimental scattering curve of 6xHis-RACK1 (open circles) and the theoretical fitting (solid line) by using the program GNOM. Inset : Guinier Region and linear regression (solid line) for R g evaluation. (B) Pair distance distribution function p(r). Inset : Kratky plot.

    Journal: BMC Structural Biology

    Article Title: Solution structure of the human signaling protein RACK1

    doi: 10.1186/1472-6807-10-15

    Figure Lengend Snippet: Small Angle X-ray Scattering (SAXS) results . (A) Experimental scattering curve of 6xHis-RACK1 (open circles) and the theoretical fitting (solid line) by using the program GNOM. Inset : Guinier Region and linear regression (solid line) for R g evaluation. (B) Pair distance distribution function p(r). Inset : Kratky plot.

    Article Snippet: Cloning of the cDNA coding for RACK1 full-length into the pET-28a plasmid (Novagen/EMD Biosciences, San Diego, CA) was performed as described previously [ ].

    Techniques:

    Sequence alignment and ab initio model for human 6xHis-RACK1 protein in solution . (A) Alignment between amino acid sequences of RACK1 from Homo sapiens (GI:83641897) versus RACK1A from A. thaliana (GI:30685669). The symbols: * showing identical residues, : residues with high similarity and residues with low similarity. (B) Experimental scattering curve of human 6xHis-RACK1 (open circles) and the calculated scattering intensity from the RACK1 from A. thaliana (solid green line) and Homo sapiens (solid red line) by using the program CRYSOL. The solid blue line represents the scattering intensity calculated from the ab initio model of 6xHis-RACK1. (C) Two orthogonal views of the homology model of human RACK1 (cartoon representation with secondary structure elements) superposed to the three dimensional low resolution ab initio model (semi-transparent yellow surface) of the human RACK1, obtained from processing the SAXS data.

    Journal: BMC Structural Biology

    Article Title: Solution structure of the human signaling protein RACK1

    doi: 10.1186/1472-6807-10-15

    Figure Lengend Snippet: Sequence alignment and ab initio model for human 6xHis-RACK1 protein in solution . (A) Alignment between amino acid sequences of RACK1 from Homo sapiens (GI:83641897) versus RACK1A from A. thaliana (GI:30685669). The symbols: * showing identical residues, : residues with high similarity and residues with low similarity. (B) Experimental scattering curve of human 6xHis-RACK1 (open circles) and the calculated scattering intensity from the RACK1 from A. thaliana (solid green line) and Homo sapiens (solid red line) by using the program CRYSOL. The solid blue line represents the scattering intensity calculated from the ab initio model of 6xHis-RACK1. (C) Two orthogonal views of the homology model of human RACK1 (cartoon representation with secondary structure elements) superposed to the three dimensional low resolution ab initio model (semi-transparent yellow surface) of the human RACK1, obtained from processing the SAXS data.

    Article Snippet: Cloning of the cDNA coding for RACK1 full-length into the pET-28a plasmid (Novagen/EMD Biosciences, San Diego, CA) was performed as described previously [ ].

    Techniques: Sequencing

    RACK1 sedimentation velocity experiments . (A) Sedimentation velocity experiments were carried out at 4°C, 40,000 rpm (AN-60Ti rotor), and with scan data acquisition at 272 nm. Data were fitted using SedFit software (Version 11.8). Figure displays the c(S) fitting for RACK1 at concentrations of 0.1, 0.2 and 0.3 mg/mL. The c(S) curves suggest that RACK1 present a predominant specie and three or four others species with higher sedimentation coefficients ( inset ) (B) Plots of s 20,ω (of the predominant specie) versus protein concentration fitted by linear regression to calculate the s 0 20;w: 3.00 ± 0.01 Svedberg.

    Journal: BMC Structural Biology

    Article Title: Solution structure of the human signaling protein RACK1

    doi: 10.1186/1472-6807-10-15

    Figure Lengend Snippet: RACK1 sedimentation velocity experiments . (A) Sedimentation velocity experiments were carried out at 4°C, 40,000 rpm (AN-60Ti rotor), and with scan data acquisition at 272 nm. Data were fitted using SedFit software (Version 11.8). Figure displays the c(S) fitting for RACK1 at concentrations of 0.1, 0.2 and 0.3 mg/mL. The c(S) curves suggest that RACK1 present a predominant specie and three or four others species with higher sedimentation coefficients ( inset ) (B) Plots of s 20,ω (of the predominant specie) versus protein concentration fitted by linear regression to calculate the s 0 20;w: 3.00 ± 0.01 Svedberg.

    Article Snippet: Cloning of the cDNA coding for RACK1 full-length into the pET-28a plasmid (Novagen/EMD Biosciences, San Diego, CA) was performed as described previously [ ].

    Techniques: Sedimentation, Software, Protein Concentration

     RACK1  hydrodynamic properties.

    Journal: BMC Structural Biology

    Article Title: Solution structure of the human signaling protein RACK1

    doi: 10.1186/1472-6807-10-15

    Figure Lengend Snippet: RACK1 hydrodynamic properties.

    Article Snippet: Cloning of the cDNA coding for RACK1 full-length into the pET-28a plasmid (Novagen/EMD Biosciences, San Diego, CA) was performed as described previously [ ].

    Techniques:

    RACK1 Sedimentation equilibrium experiments . (A) The figure shows the best fits of experimental data for 300 μg/mL of RACK1 (4 °C) at 12000, 15000, and 18000 rpm with the self-association methods (SedPhat program - Material and Methods). The random distribution of the residuals (bottom panel) indicates that the fit is satisfactory. Sedimentation equilibrium data of RACK1 agree with a monomer structure with 37.2 kDa in equilibrium with of dimers and tetramers (see Results and Discussion for details). (B) Sedimentation equilibrium interaction between RACK1 and KI-1/57(122-413) was tested at 300 μg/mL of both proteins at 12,000, 15,000, and 18,000 rpm and 4 °C. The data were fitted by self-association method (SedPhat program - Material and Methods) and resulted in random distribution of the residuals (bottom panel) which indicates that the fit is satisfactory. The fitting data suggest that RACK1 and Ki-1/57(122-413) interacts with an affinity constant of (1.5 ± 0.2) × 10 6 M -1 .

    Journal: BMC Structural Biology

    Article Title: Solution structure of the human signaling protein RACK1

    doi: 10.1186/1472-6807-10-15

    Figure Lengend Snippet: RACK1 Sedimentation equilibrium experiments . (A) The figure shows the best fits of experimental data for 300 μg/mL of RACK1 (4 °C) at 12000, 15000, and 18000 rpm with the self-association methods (SedPhat program - Material and Methods). The random distribution of the residuals (bottom panel) indicates that the fit is satisfactory. Sedimentation equilibrium data of RACK1 agree with a monomer structure with 37.2 kDa in equilibrium with of dimers and tetramers (see Results and Discussion for details). (B) Sedimentation equilibrium interaction between RACK1 and KI-1/57(122-413) was tested at 300 μg/mL of both proteins at 12,000, 15,000, and 18,000 rpm and 4 °C. The data were fitted by self-association method (SedPhat program - Material and Methods) and resulted in random distribution of the residuals (bottom panel) which indicates that the fit is satisfactory. The fitting data suggest that RACK1 and Ki-1/57(122-413) interacts with an affinity constant of (1.5 ± 0.2) × 10 6 M -1 .

    Article Snippet: Cloning of the cDNA coding for RACK1 full-length into the pET-28a plasmid (Novagen/EMD Biosciences, San Diego, CA) was performed as described previously [ ].

    Techniques: Sedimentation

    Titration of Ki-1/57(122-413) into RACK1 solution monitored by fluorescence . (A) Fluorescence emission spectra titration of purified 6xHis RACK1 protein (1 μM) with titration of Ki-1/57 at 0.1 μM (black, filled square), 0.2 μM (black, filled circle), 0.3 μM (black, filled triangle), 0.5 μM (open, white triangle) and 1 μM (open, white circle). The excitation wavelength was 295 nm and spectra were background corrected. Inset : Area under curve (AUC) of RACK1 fluorescence emission spectrum in function of Ki-1/57(122-413) concentration. The arrow indicates the increase on the RACK1 fluorescence signal at Ki-1/57(122-413) 0.3 uM. ( B ) The titration curve was represented as difference of fluorescence intensity at 334 nm θ (black, filled square) as function of Ki-1/57(122-413) concentration in the range from 0.3 μM and 1.4 μM and fitted with the modified Hill equation (Equation 5). The dashed line represents the fitting curve. The KD between RACK1 and Ki-1/57(122-413) was of around 0.60 μM which corresponds to the dissociation constant of RACK1\Ki-1/57(122-413) observed in sedimentation equilibrium experiments. The number of cooperative sites n was 8 ± 1.

    Journal: BMC Structural Biology

    Article Title: Solution structure of the human signaling protein RACK1

    doi: 10.1186/1472-6807-10-15

    Figure Lengend Snippet: Titration of Ki-1/57(122-413) into RACK1 solution monitored by fluorescence . (A) Fluorescence emission spectra titration of purified 6xHis RACK1 protein (1 μM) with titration of Ki-1/57 at 0.1 μM (black, filled square), 0.2 μM (black, filled circle), 0.3 μM (black, filled triangle), 0.5 μM (open, white triangle) and 1 μM (open, white circle). The excitation wavelength was 295 nm and spectra were background corrected. Inset : Area under curve (AUC) of RACK1 fluorescence emission spectrum in function of Ki-1/57(122-413) concentration. The arrow indicates the increase on the RACK1 fluorescence signal at Ki-1/57(122-413) 0.3 uM. ( B ) The titration curve was represented as difference of fluorescence intensity at 334 nm θ (black, filled square) as function of Ki-1/57(122-413) concentration in the range from 0.3 μM and 1.4 μM and fitted with the modified Hill equation (Equation 5). The dashed line represents the fitting curve. The KD between RACK1 and Ki-1/57(122-413) was of around 0.60 μM which corresponds to the dissociation constant of RACK1\Ki-1/57(122-413) observed in sedimentation equilibrium experiments. The number of cooperative sites n was 8 ± 1.

    Article Snippet: Cloning of the cDNA coding for RACK1 full-length into the pET-28a plasmid (Novagen/EMD Biosciences, San Diego, CA) was performed as described previously [ ].

    Techniques: Titration, Fluorescence, Purification, Concentration Assay, Modification, Sedimentation

    RACK1 is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: RACK1 is highly expressed in CC cell lines and regulates CC cell migration and invasion in vitro . (A) Protein levels of RACK1 in CC cell lines (MS751, HeLa, Caski and SiHa) and normal cell line (H8) detected using western blot analysis (left panel), with the protein bands assessed (right panel). (B) RACK1 mRNA level in cell lines (MS751, HeLa, Caski, SiHa and H8) detected using RT-qPCR. (C) RACK1 expression in MS751 and SiHa cells transfected with specific shRACK1 lentiviral vectors (shRACK1-1 and shRACK1-2) was examined using western blot analysis and (D) RT-qPCR. (E) Transwell assays were performed to investigate the effects of RACK1 on the invasion and migration of MS751 cells (left panel), with the quantified bands assessed (right panel). (F) Transwell assays were performed to investigate the effects of RACK1 on the migration ability of HLECs (left panel), with the quantified bands assessed (right panel). (G) Effects of RACK1 on tube formation by HLECs. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the the H8 cell line or the shNON group ( * P<0.05 and *** P<0.001). CC, cervical cancer; RACK1, receptor for activated C kinase 1; HLECs, human lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: Migration, In Vitro, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Real-time Polymerase Chain Reaction

    RACK1 enhances the glycolysis in cervical cancer cells. (A) Common differential metabolites in the metabolic pathway of the supernatant of shRACK1/MS751 and shRACK1/SiHa cells. Aminoacyl-tRNA biosynthesis; Valine, leucine and isoleucine biosynthesis; glycolysis/gluconeogenesis; Glyoxylate and dicarboxylate metabolism; glycine, serine and threonine metabolism; arginine and proline metabolism; valine, leucine and isoleucine degradation; butanoate metabolism; pyruvate metabolism; glutathione metabolism. (B) Effect of RACK1 on glucose uptake and lactate production. (C) Effect of RACK1 on the expression of the AKT/mTOR pathway, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using western blot analysis (left panel), with the protein bands assessed (right panel). (D) Effect of RACK1 on the mRNA expression of the GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using reverse transcription-quantitative PCR analysis. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shNON group ( * P<0.05, ** P<0.01 and *** P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: RACK1 enhances the glycolysis in cervical cancer cells. (A) Common differential metabolites in the metabolic pathway of the supernatant of shRACK1/MS751 and shRACK1/SiHa cells. Aminoacyl-tRNA biosynthesis; Valine, leucine and isoleucine biosynthesis; glycolysis/gluconeogenesis; Glyoxylate and dicarboxylate metabolism; glycine, serine and threonine metabolism; arginine and proline metabolism; valine, leucine and isoleucine degradation; butanoate metabolism; pyruvate metabolism; glutathione metabolism. (B) Effect of RACK1 on glucose uptake and lactate production. (C) Effect of RACK1 on the expression of the AKT/mTOR pathway, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using western blot analysis (left panel), with the protein bands assessed (right panel). (D) Effect of RACK1 on the mRNA expression of the GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin, as evaluated using reverse transcription-quantitative PCR analysis. All data were obtained from three independent experiments. The data of two groups were analyzed using the Student's t-test and the data of more than two groups were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shNON group ( * P<0.05, ** P<0.01 and *** P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    RACK1 interacts with IGF1R promotes the glycolysis, aggressiveness and lymphangiogenesis by activating IGF1R/AKT/mTOR signaling in MS751 cells. (A) Venn diagram showing the factors interacting with RACK1, co-existing in the Hitpredict and Genemania databases. (B) Association between RACK1 and IGF1R expression was analyzed using immunofluorescence staining in MS751 and SiHa cells. Nuclei were stained with DAPI (blue). (C) Co-IP assays were used to detect the interaction between RACK1 and IGF1R in MS751 cells (upper panel). Co-IP assay was used to detect the interaction between RACK1 and IGF1R in shRACK1/MS751 and shNON/MS751 cells (lower panel). (D) shRACK1/MS751 cells were stimulated for different time periods (6, 12 and 24 h) with 200 ng/ml IGF1. The RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2 and LDHA, E-cadherin and N-cadherin expression was examined using western blot analysis. (E) shRACK1/MS751 cells were stimulated for 24 h with 200 ng/ml IGF1 and various concentrations (10 and 20 nM) of Rapa. The expression of RACK1, p-mTOR (ser2448), mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin was examined using western blot analysis. (F) Transwell assays were performed to investigate the effects of IGF1 and IGF1 combined with Rapa on the invasion and migration of MS751 and HLECs cells (upper panel), with the quantified bands assessed (lower panel). (G) Effect of IGF1 and IGF1 combined with Rapa on tube formation by HLECs. (H) Effect of IGF1 and IGF1 combined with Rapa on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( * P<0.05 and *** P<0.001). or shRACK1 + IGF1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2; Rapa, rapamycin; IGF1, insulin-like growth factor 1; HLECs, human lymphatic endothelial cells.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: RACK1 interacts with IGF1R promotes the glycolysis, aggressiveness and lymphangiogenesis by activating IGF1R/AKT/mTOR signaling in MS751 cells. (A) Venn diagram showing the factors interacting with RACK1, co-existing in the Hitpredict and Genemania databases. (B) Association between RACK1 and IGF1R expression was analyzed using immunofluorescence staining in MS751 and SiHa cells. Nuclei were stained with DAPI (blue). (C) Co-IP assays were used to detect the interaction between RACK1 and IGF1R in MS751 cells (upper panel). Co-IP assay was used to detect the interaction between RACK1 and IGF1R in shRACK1/MS751 and shNON/MS751 cells (lower panel). (D) shRACK1/MS751 cells were stimulated for different time periods (6, 12 and 24 h) with 200 ng/ml IGF1. The RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2 and LDHA, E-cadherin and N-cadherin expression was examined using western blot analysis. (E) shRACK1/MS751 cells were stimulated for 24 h with 200 ng/ml IGF1 and various concentrations (10 and 20 nM) of Rapa. The expression of RACK1, p-mTOR (ser2448), mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin was examined using western blot analysis. (F) Transwell assays were performed to investigate the effects of IGF1 and IGF1 combined with Rapa on the invasion and migration of MS751 and HLECs cells (upper panel), with the quantified bands assessed (lower panel). (G) Effect of IGF1 and IGF1 combined with Rapa on tube formation by HLECs. (H) Effect of IGF1 and IGF1 combined with Rapa on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( * P<0.05 and *** P<0.001). or shRACK1 + IGF1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; mTOR, mammalian target of rapamycin; GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2; Rapa, rapamycin; IGF1, insulin-like growth factor 1; HLECs, human lymphatic endothelial cells.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: Expressing, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Migration

    RACK1 is a direct target of POU2F2. (A) Venn diagram showing the transcription factor binding to RACK1 co-existing in UCSC, HUMANTFDB and PROMO database. (B) POU2F2 DNA binding sites are present in the human RACK1 promoter region. The top panel shows the WT and MUT forms of the putative POU2F2 target sequences in RACK1 3′-UTR. Red font (upper panel) refers to the putative POU2F2 targeting sequence in the RACK1 3′-UTR. Red font (lower panel) refers to mutations in the POU2F2 targeting sequence in RACK1 3′-UTR. (C) Luciferase reporter assays of WT and MUT RACK1 luciferase reporters transfected with POU2F2 in MS751 cells. (D) ChIP-PCR assay was used to detect POU2F2-binding sites in the sequence of the RACK1 promoter. (E) Effect of POU2F2 on the expression of RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin in shRACK1/MS751 cells, as evaluated using western blot analysis. (F) POU2F2 OE-shRACK1/MS751 cells were stimulated for 24 h with 10 mM 2-DG. Effect of POU2F2 and 2-DG combined with POU2F2 on the invasion and migration of shRACK1/MS751 cells and migration of HLECs (upper panel), with the quantified bands assessed (lower panel). (G) Effect of POU2F2 on tube formation of HLECs. (H) Effect of POU2F2 on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( ** P<0.01 and *** P<0.001) or OE POU2F2/shRACK1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; POU2F2, POU class 2 homeobox 2; WT, wild-type; MUT, mutant type; 2-DG, 2-deoxy-D-glucose; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; HLECs, human lymphatic endothelial cells; OE, overexpression.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: RACK1 is a direct target of POU2F2. (A) Venn diagram showing the transcription factor binding to RACK1 co-existing in UCSC, HUMANTFDB and PROMO database. (B) POU2F2 DNA binding sites are present in the human RACK1 promoter region. The top panel shows the WT and MUT forms of the putative POU2F2 target sequences in RACK1 3′-UTR. Red font (upper panel) refers to the putative POU2F2 targeting sequence in the RACK1 3′-UTR. Red font (lower panel) refers to mutations in the POU2F2 targeting sequence in RACK1 3′-UTR. (C) Luciferase reporter assays of WT and MUT RACK1 luciferase reporters transfected with POU2F2 in MS751 cells. (D) ChIP-PCR assay was used to detect POU2F2-binding sites in the sequence of the RACK1 promoter. (E) Effect of POU2F2 on the expression of RACK1, p-AKT (ser472 + ser474 + ser473), p-mTOR (ser2448), AKT, mTOR, GLUT1, HK2, PKM2, LDHA, E-cadherin and N-cadherin in shRACK1/MS751 cells, as evaluated using western blot analysis. (F) POU2F2 OE-shRACK1/MS751 cells were stimulated for 24 h with 10 mM 2-DG. Effect of POU2F2 and 2-DG combined with POU2F2 on the invasion and migration of shRACK1/MS751 cells and migration of HLECs (upper panel), with the quantified bands assessed (lower panel). (G) Effect of POU2F2 on tube formation of HLECs. (H) Effect of POU2F2 on glucose uptake and lactate production in MS751 cells. Data were analyzed using one-way ANOVA and are presented as the mean ± SD. Data were compared with the shRACK1 group ( ** P<0.01 and *** P<0.001) or OE POU2F2/shRACK1 group ( # P<0.05 and ### P<0.001). RACK1, receptor for activated C kinase 1; POU2F2, POU class 2 homeobox 2; WT, wild-type; MUT, mutant type; 2-DG, 2-deoxy-D-glucose; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; HLECs, human lymphatic endothelial cells; OE, overexpression.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: Binding Assay, Sequencing, Luciferase, Transfection, Expressing, Western Blot, Migration, Mutagenesis, Over Expression

    RACK1 enhances the LNM of MS751 cells in vivo . (A) Representative images of inguinal lymph nodes in different groups of nude mice (n=8, left panel). Representative images of inguinal lymph nodes of H&E staining in different groups of nude mice (middle panel). Representative images of anti-GFP IHC analysis for inguinal lymph nodes in different groups of nude mice (right panel). (B) Representative images of footpads primary tumor in different groups of nude mice (left panel). Representative images of footpads primary tumor tissues of H&E staining (middle panel) and percentages of PDPN-indicated lymphatic vessels density in different groups of nude mice (right panel). (C) The image of all tumors with a ruler. (D) Representative images of RACK1, GLUT1, PKM2, HK2, LDHA, E-cadherin and N-cadherin expression in footpad primary tumor tissues in IHC analysis. (E) The bar graph summarizes the tumor size assessed (left panel), and lymph node volume assessed (right panel). Data were compared with the shNON group ( ** P<0.01 and *** P<0.001). Data are presented as the mean ± SD and were analyzed using the t-test. IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; PDPN, podoplanin.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: RACK1 enhances the LNM of MS751 cells in vivo . (A) Representative images of inguinal lymph nodes in different groups of nude mice (n=8, left panel). Representative images of inguinal lymph nodes of H&E staining in different groups of nude mice (middle panel). Representative images of anti-GFP IHC analysis for inguinal lymph nodes in different groups of nude mice (right panel). (B) Representative images of footpads primary tumor in different groups of nude mice (left panel). Representative images of footpads primary tumor tissues of H&E staining (middle panel) and percentages of PDPN-indicated lymphatic vessels density in different groups of nude mice (right panel). (C) The image of all tumors with a ruler. (D) Representative images of RACK1, GLUT1, PKM2, HK2, LDHA, E-cadherin and N-cadherin expression in footpad primary tumor tissues in IHC analysis. (E) The bar graph summarizes the tumor size assessed (left panel), and lymph node volume assessed (right panel). Data were compared with the shNON group ( ** P<0.01 and *** P<0.001). Data are presented as the mean ± SD and were analyzed using the t-test. IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; PDPN, podoplanin.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: In Vivo, Staining, Expressing, Immunohistochemistry

     RACK1  expression in cervical carcinoma according to the histopathological characteristics of the patients with cervical cancer.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: RACK1 expression in cervical carcinoma according to the histopathological characteristics of the patients with cervical cancer.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: Expressing

    Associations between RACK1, IGF1R, POU2F2, and HK2 expression in tissues from patients with CC. Representative images of IHC staining in case 1 for RACK1, IGF1R, POU2F2 and HK2 (strong RACK1 expression). Representative images of IHC staining in case 2 for RACK1, IGF1R, POU2F2 and HK2 (medium RACK1 expression). Representative images of IHC staining in case 3 for RACK1, IGF1R, POU2F2 and HK2 (weak RACK1 expression). Representative images of IHC staining in case 4 for RACK1, IGF1R, POU2F2 and HK2 (negative RACK1 expression). CC, cervical cancer; IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: Associations between RACK1, IGF1R, POU2F2, and HK2 expression in tissues from patients with CC. Representative images of IHC staining in case 1 for RACK1, IGF1R, POU2F2 and HK2 (strong RACK1 expression). Representative images of IHC staining in case 2 for RACK1, IGF1R, POU2F2 and HK2 (medium RACK1 expression). Representative images of IHC staining in case 3 for RACK1, IGF1R, POU2F2 and HK2 (weak RACK1 expression). Representative images of IHC staining in case 4 for RACK1, IGF1R, POU2F2 and HK2 (negative RACK1 expression). CC, cervical cancer; IHC, immunohistochemistry; RACK1, receptor for activated C kinase 1; LNM, lymph node metastasis; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: Expressing, Immunohistochemistry

    Schematic illustration of the mechanisms by which the POU2F2/RACK1/IGF1R/AKT/mTOR pathway promotes cell lymph node metastasis dependent on glycolysis. RACK1, receptor for activated C kinase 1; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2.

    Journal: International Journal of Oncology

    Article Title: Receptor for activated C kinase 1 promotes cervical cancer lymph node metastasis via the glycolysis-dependent AKT/mTOR signaling

    doi: 10.3892/ijo.2022.5373

    Figure Lengend Snippet: Schematic illustration of the mechanisms by which the POU2F2/RACK1/IGF1R/AKT/mTOR pathway promotes cell lymph node metastasis dependent on glycolysis. RACK1, receptor for activated C kinase 1; IGF1R, insulin-like growth factor 1 receptor; POU2F2, POU class 2 homeobox 2; mTOR, mammalian target of rapamycin; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; GLUT1, glucose transporter 1; PKM2, pyruvate kinase M2.

    Article Snippet: The cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 ( ) at room temperature for 2 h. The coverslips were then incubated with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG ( ) at room temperature for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for at room temperature for 10 min. Randomized fields were imaged using a confocal laser microscope (LSM 980, Zeiss AG).

    Techniques: