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Proteintech rabbit fbxw2

<t>FBXW2</t> is down-regulated in highly-metastasis PCa cells and tissues, and enhanced FBXW2 expression inhibits cancer growth and metastasis. a Both mRNA and protein levels of FBXW2 were significantly decreased in highly-progressive PCa tissues. We collected 9 clinical samples and divided them into three groups: prostatic hyperplasia (Nor), Non-metastatic PCa (PCa, Gleason score ≤ 7) and metastatic prostate cancer (M-PCa, Gleason score > 7). Then, the mRNA and protein levels of FBXW2 in clinical samples were analyzed by qPCR analysis and immunoblotting (IB), respectively ( *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). b FBXW2 protein levels in prostate cell lines were analyzed by IB analysis (ns, no significance, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). c – e Overexpression of FBXW2 in PC3 cells inhibited cell proliferation ( c ), invasion ( d ), and migration ( e ). Scale bar:100 µm ( d ) and 200 µm ( e ). Cell viability was detected with Cell Counting Kit-8 (CCK-8) assay ( ***p < 0.001; n = 3; two-way ANOVA). The cell migration ability and invasion ability were determined using the wound-healing and transwell assays, respectively ( ***p < 0.001; n = 3; Student’s two-tailed t test). f Overexpression of FBXW2 induced G1-phase cell cycle arrest in PC3 cells. The PC3 cells were transfected with vector or HA-FBXW2, and cell cycle distributions were then analyzed by flow cytometry ( **p < 0.01, ***p < 0.001; n = 3; Student’s two-tailed t test). g Effects of FBXW2 overexpression on the expression of cell progression-related proteins in PC3 cells. GAPDH levels served as the control for equal loading ( ***p < 0.001; n = 3; Student’s two-tailed t test)

Rabbit Fbxw2, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+fbxw2/FBXW2+Antibody/pmc09061686-175-13-15
Average 93 stars, based on 8 article reviews

rabbit fbxw2 - by Bioz Stars, 2026-10

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1) Product Images from "FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation"

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

Journal: Cellular and Molecular Life Sciences

doi: 10.1007/s00018-022-04320-3

FBXW2 is down-regulated in highly-metastasis PCa cells and tissues, and enhanced FBXW2 expression inhibits cancer growth and metastasis. a Both mRNA and protein levels of FBXW2 were significantly decreased in highly-progressive PCa tissues. We collected 9 clinical samples and divided them into three groups: prostatic hyperplasia (Nor), Non-metastatic PCa (PCa, Gleason score ≤ 7) and metastatic prostate cancer (M-PCa, Gleason score > 7). Then, the mRNA and protein levels of FBXW2 in clinical samples were analyzed by qPCR analysis and immunoblotting (IB), respectively ( *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). b FBXW2 protein levels in prostate cell lines were analyzed by IB analysis (ns, no significance, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). c – e Overexpression of FBXW2 in PC3 cells inhibited cell proliferation ( c ), invasion ( d ), and migration ( e ). Scale bar:100 µm ( d ) and 200 µm ( e ). Cell viability was detected with Cell Counting Kit-8 (CCK-8) assay ( ***p < 0.001; n = 3; two-way ANOVA). The cell migration ability and invasion ability were determined using the wound-healing and transwell assays, respectively ( ***p < 0.001; n = 3; Student’s two-tailed t test). f Overexpression of FBXW2 induced G1-phase cell cycle arrest in PC3 cells. The PC3 cells were transfected with vector or HA-FBXW2, and cell cycle distributions were then analyzed by flow cytometry ( **p < 0.01, ***p < 0.001; n = 3; Student’s two-tailed t test). g Effects of FBXW2 overexpression on the expression of cell progression-related proteins in PC3 cells. GAPDH levels served as the control for equal loading ( ***p < 0.001; n = 3; Student’s two-tailed t test)

Figure Legend Snippet: FBXW2 is down-regulated in highly-metastasis PCa cells and tissues, and enhanced FBXW2 expression inhibits cancer growth and metastasis. a Both mRNA and protein levels of FBXW2 were significantly decreased in highly-progressive PCa tissues. We collected 9 clinical samples and divided them into three groups: prostatic hyperplasia (Nor), Non-metastatic PCa (PCa, Gleason score ≤ 7) and metastatic prostate cancer (M-PCa, Gleason score > 7). Then, the mRNA and protein levels of FBXW2 in clinical samples were analyzed by qPCR analysis and immunoblotting (IB), respectively ( *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). b FBXW2 protein levels in prostate cell lines were analyzed by IB analysis (ns, no significance, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). c – e Overexpression of FBXW2 in PC3 cells inhibited cell proliferation ( c ), invasion ( d ), and migration ( e ). Scale bar:100 µm ( d ) and 200 µm ( e ). Cell viability was detected with Cell Counting Kit-8 (CCK-8) assay ( ***p < 0.001; n = 3; two-way ANOVA). The cell migration ability and invasion ability were determined using the wound-healing and transwell assays, respectively ( ***p < 0.001; n = 3; Student’s two-tailed t test). f Overexpression of FBXW2 induced G1-phase cell cycle arrest in PC3 cells. The PC3 cells were transfected with vector or HA-FBXW2, and cell cycle distributions were then analyzed by flow cytometry ( **p < 0.01, ***p < 0.001; n = 3; Student’s two-tailed t test). g Effects of FBXW2 overexpression on the expression of cell progression-related proteins in PC3 cells. GAPDH levels served as the control for equal loading ( ***p < 0.001; n = 3; Student’s two-tailed t test)

Techniques Used: Expressing, Western Blot, Over Expression, Migration, Cell Counting, CCK-8 Assay, Two Tailed Test, Transfection, Plasmid Preparation, Flow Cytometry, Control

Augmented FBXW2 inhibits PCa tumor growth and attenuates osteolytic bone tumor tumorigenesis. a–c Overexpression of FBXW2 inhibited PCa tumor growth in vivo. PC3 cells stably expressing Vector and HA-FBXW2 (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. After indicated time, the tumors were harvested and photographed ( a ) ( ***p < 0.001; n = 5; Student’s two-tailed t test; Scale bars, 200 μm). The tumor growth was monitored twice a week for up to 30 days and growth curve plotted ( b ) ( ***p < 0.001; n = 5; two-way ANOVA). Body weight was measured and plotted ( c ) (ns, no significance; n = 5; two-way ANOVA). d Overexpression of FBXW2 attenuated osteolytic bone tumor tumorigenesis in vivo. 2.0 × 10 6 PC3 stable cells (Vector or HA-FBXW2) were re-suspended in 100 µL PBS, and 10 µL of cell solution was slowly injected into NOD/SCID mice. Representative radiographical images of osteolytic bone tumor in the indicated tibia of the mice (left). Bars, 4 mm. Representative H&E-stained sections of the indicated tibia of the mice (right). Scale bar, 500 µm and 50 µm. The sum of bone metastasis scores in the tibia of the Vector or HA-FBXW2 mice groups ( ***p < 0.001; n = 10; Student’s two-tailed t test)

Figure Legend Snippet: Augmented FBXW2 inhibits PCa tumor growth and attenuates osteolytic bone tumor tumorigenesis. a–c Overexpression of FBXW2 inhibited PCa tumor growth in vivo. PC3 cells stably expressing Vector and HA-FBXW2 (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. After indicated time, the tumors were harvested and photographed ( a ) ( ***p < 0.001; n = 5; Student’s two-tailed t test; Scale bars, 200 μm). The tumor growth was monitored twice a week for up to 30 days and growth curve plotted ( b ) ( ***p < 0.001; n = 5; two-way ANOVA). Body weight was measured and plotted ( c ) (ns, no significance; n = 5; two-way ANOVA). d Overexpression of FBXW2 attenuated osteolytic bone tumor tumorigenesis in vivo. 2.0 × 10 6 PC3 stable cells (Vector or HA-FBXW2) were re-suspended in 100 µL PBS, and 10 µL of cell solution was slowly injected into NOD/SCID mice. Representative radiographical images of osteolytic bone tumor in the indicated tibia of the mice (left). Bars, 4 mm. Representative H&E-stained sections of the indicated tibia of the mice (right). Scale bar, 500 µm and 50 µm. The sum of bone metastasis scores in the tibia of the Vector or HA-FBXW2 mice groups ( ***p < 0.001; n = 10; Student’s two-tailed t test)

Techniques Used: Over Expression, In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Two Tailed Test, Injection, Staining

FBXW2 is inversely correlated with EGFR in PCa, and regulates EGFR protein level. a Inverse correlation at the protein levels of EGFR versus FBXW2 in PCa cell lines ( *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). b Expression of FBXW2 and EGFR in PCa tissues. PCa tissue microarrays were stained with FBXW2 and EGFR, and then photographed (Scale bars, 100 μm). Association analysis of FBXW2 and EGFR in PCa tissues. Data were analyzed using SPSS software ( P = 0.001, n = 42, Pearson’s test). c , d Overexpression of FBXW2 reduced EGFR protein level, but not EGFR mRNA levels. PC3 cells were transfected with vector or HA-FBXW2, and followed by IB ( c ) or qPCR ( d ) ( ***p < 0.001; n = 3; Student’s two-tailed t test). e FBXW2 could bind to endogenous EGFR: Cell lysates from PC3 cells were pulled down with anti-FBXW2 Abs, followed by IB with indicated Abs. f Overexpression of FBXW2, but not its ΔF mutant, shortened protein half-life of exogenous EGFR. After transfection with relevant plasmids for 48 h, 293 T cells were switched to fresh medium (10% FBS) containing cycloheximide (CHX) and incubated for indicated time periods before being harvested for IB. The band density was quantified using ImageJ software and plotted ( **p < 0.01; ***p < 0.001; n = 3; two-way ANOVA). g Protein levels of EGFR downstream were markedly decreased by FBXW2 overexpression. PC3 cells were transfected with vector or HA-FBXW2, and followed by IB (ns, no significance, ***p < 0.001; n = 3; Student’s two-tailed t test). h , i Transfection of FBXW2 significantly abrogated invasion ability ( h ) and proliferation ( i ) caused by EGF. Scale bar: 100 µm ( h ). Transfected the vector control or plasmid expressing HA-FBXW2 for 48 h in PC3 cells were treated with or without EGF (10 ng/mL). The cell ability and invasion ability were detected by CCK-8 assay ( *p < 0.1, ***p < 0.001; n = 3; two-way ANOVA) and transwell assays (* **p < 0.001; n = 3; one-way ANOVA), respectively

Figure Legend Snippet: FBXW2 is inversely correlated with EGFR in PCa, and regulates EGFR protein level. a Inverse correlation at the protein levels of EGFR versus FBXW2 in PCa cell lines ( *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). b Expression of FBXW2 and EGFR in PCa tissues. PCa tissue microarrays were stained with FBXW2 and EGFR, and then photographed (Scale bars, 100 μm). Association analysis of FBXW2 and EGFR in PCa tissues. Data were analyzed using SPSS software ( P = 0.001, n = 42, Pearson’s test). c , d Overexpression of FBXW2 reduced EGFR protein level, but not EGFR mRNA levels. PC3 cells were transfected with vector or HA-FBXW2, and followed by IB ( c ) or qPCR ( d ) ( ***p < 0.001; n = 3; Student’s two-tailed t test). e FBXW2 could bind to endogenous EGFR: Cell lysates from PC3 cells were pulled down with anti-FBXW2 Abs, followed by IB with indicated Abs. f Overexpression of FBXW2, but not its ΔF mutant, shortened protein half-life of exogenous EGFR. After transfection with relevant plasmids for 48 h, 293 T cells were switched to fresh medium (10% FBS) containing cycloheximide (CHX) and incubated for indicated time periods before being harvested for IB. The band density was quantified using ImageJ software and plotted ( **p < 0.01; ***p < 0.001; n = 3; two-way ANOVA). g Protein levels of EGFR downstream were markedly decreased by FBXW2 overexpression. PC3 cells were transfected with vector or HA-FBXW2, and followed by IB (ns, no significance, ***p < 0.001; n = 3; Student’s two-tailed t test). h , i Transfection of FBXW2 significantly abrogated invasion ability ( h ) and proliferation ( i ) caused by EGF. Scale bar: 100 µm ( h ). Transfected the vector control or plasmid expressing HA-FBXW2 for 48 h in PC3 cells were treated with or without EGF (10 ng/mL). The cell ability and invasion ability were detected by CCK-8 assay ( *p < 0.1, ***p < 0.001; n = 3; two-way ANOVA) and transwell assays (* **p < 0.001; n = 3; one-way ANOVA), respectively

Techniques Used: Expressing, Staining, Software, Over Expression, Transfection, Plasmid Preparation, Two Tailed Test, Mutagenesis, Incubation, Control, CCK-8 Assay

FBXW2 binds to EGFR via its consensus degron motif, and degrades EGFR. a Two possible binding sites on EGFR, MU1 (446/447, 452) and MU2 (1041/1042, 1045/1046). b Loss of FBXW2-EGFR binding site in mutant: EGFR or its mutants was co-transfected with HA-FBXW2, followed by IP with HA Abs and IB. c Overexpression of FBXW2 reduced basal level of EGFR and EGFR-MU1 in a dose manner, but not EGFR-MU2. (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). d FBXW2 shortened the protein half-life of EGFR and EGFR-MU1, but not EGFR-MU2. Stable 293 T cells were incubated with CHX for indicated time periods and harvested for IB (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; two-way ANOVA). e CK1 kinase mediated EGFR phosphorylation at FBXW2 binding motif. PC3 cells were transfected HA-FBXW2, and treated with or without CK1 inhibitor IC261 (10 mM), and harvested for IB. GAPDH levels served as the control for equal loading (ns, no significance, ***p < 0.001; n = 3; two-way ANOVA). f , g Under EGF stimulation, transfected FBXW2 alone or co-transfection with wild-type EGFR suppressed and invasion ( f ) and cell proliferation ( g ) in PC3 cells, but was abrogated by simultaneous transfection of EGFR-MU2. Scale bar:100 µm ( f ). Transfected HA-FBXW2 alone or in combinations with FLAG-EGFR (WT versus MU2) into PC3 cells and then treated with EGF (10 ng/mL). The cell proliferation and invasion ability were detected by CCK-8 assay ( **p < 0.01, ***p < 0.001; n = 3; two-way ANOVA) and transwell assays ( **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA), respectively

Figure Legend Snippet: FBXW2 binds to EGFR via its consensus degron motif, and degrades EGFR. a Two possible binding sites on EGFR, MU1 (446/447, 452) and MU2 (1041/1042, 1045/1046). b Loss of FBXW2-EGFR binding site in mutant: EGFR or its mutants was co-transfected with HA-FBXW2, followed by IP with HA Abs and IB. c Overexpression of FBXW2 reduced basal level of EGFR and EGFR-MU1 in a dose manner, but not EGFR-MU2. (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). d FBXW2 shortened the protein half-life of EGFR and EGFR-MU1, but not EGFR-MU2. Stable 293 T cells were incubated with CHX for indicated time periods and harvested for IB (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; two-way ANOVA). e CK1 kinase mediated EGFR phosphorylation at FBXW2 binding motif. PC3 cells were transfected HA-FBXW2, and treated with or without CK1 inhibitor IC261 (10 mM), and harvested for IB. GAPDH levels served as the control for equal loading (ns, no significance, ***p < 0.001; n = 3; two-way ANOVA). f , g Under EGF stimulation, transfected FBXW2 alone or co-transfection with wild-type EGFR suppressed and invasion ( f ) and cell proliferation ( g ) in PC3 cells, but was abrogated by simultaneous transfection of EGFR-MU2. Scale bar:100 µm ( f ). Transfected HA-FBXW2 alone or in combinations with FLAG-EGFR (WT versus MU2) into PC3 cells and then treated with EGF (10 ng/mL). The cell proliferation and invasion ability were detected by CCK-8 assay ( **p < 0.01, ***p < 0.001; n = 3; two-way ANOVA) and transwell assays ( **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA), respectively

Techniques Used: Binding Assay, Mutagenesis, Transfection, Over Expression, Incubation, Phospho-proteomics, Control, Cotransfection, CCK-8 Assay

Figure Legend Snippet: Putative kinases for FBXW2 phosphorylation at the EGFR binding motif (TSNNST)

Techniques Used: Phospho-proteomics, Binding Assay

FBXW2 promotes EGFR ubiquitylation via its consensus degron motif (TSNNST) on EGFR for subsequent degradation. a FBXW2 promoted ubiquitylation of EGFR in vivo: PC3 cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. b FBXW2, but not its ΔF mutant, promoted ubiquitylation of EGFR in vivo: 293 T cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. c EGFR was conjugated with lysine (K) 48- or 63-linked polyubiquitin chains: 293 T cells transfected with different ubiquitin mutants were co-overexpressed both HA-FBXW2 and FLAG-EGFR. After 48 h transfection, cells were treated with MG132 for 4 h followed by IP and IB. d FBXW2 promoted ubiquitylation of wild-type EGFR and EGFR-MU1, not EGFR-MU2 in vivo: 293 T cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. GAPDH levels served as the control for equal loading

Figure Legend Snippet: FBXW2 promotes EGFR ubiquitylation via its consensus degron motif (TSNNST) on EGFR for subsequent degradation. a FBXW2 promoted ubiquitylation of EGFR in vivo: PC3 cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. b FBXW2, but not its ΔF mutant, promoted ubiquitylation of EGFR in vivo: 293 T cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. c EGFR was conjugated with lysine (K) 48- or 63-linked polyubiquitin chains: 293 T cells transfected with different ubiquitin mutants were co-overexpressed both HA-FBXW2 and FLAG-EGFR. After 48 h transfection, cells were treated with MG132 for 4 h followed by IP and IB. d FBXW2 promoted ubiquitylation of wild-type EGFR and EGFR-MU1, not EGFR-MU2 in vivo: 293 T cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. GAPDH levels served as the control for equal loading

Techniques Used: In Vivo, Transfection, Mutagenesis, Ubiquitin Proteomics, Control

Schematic model for FBXW2-induced EGFR degradation inhibiting growth and metastasis of PCa cells. During tumorigenesis, FBXW2, as a tumor suppressor, promotes ubiquitylation and degradation of EGFR, leading to repression of EGFR downstream, eventually to control growth and metastasis of PCa cells

Figure Legend Snippet: Schematic model for FBXW2-induced EGFR degradation inhibiting growth and metastasis of PCa cells. During tumorigenesis, FBXW2, as a tumor suppressor, promotes ubiquitylation and degradation of EGFR, leading to repression of EGFR downstream, eventually to control growth and metastasis of PCa cells

Techniques Used: Control

Expressing:

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation
Article Snippet: For direct IB analysis, cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors (Roche Life Science, Switzerland).. The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).. For immune-precipitation, cells were treated by MG132 (10 μg/mL, Beyotime, China) for 4 h before lysed in IP-RIPA buffer with protease inhibitors, and Protein A/G plus Agarose (Santa Cruz, USA) were used to pull down the proteins.

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation
Article Snippet: 2.8 Immunoblotting (IB) and Immunoprecipitation (IP) For direct IB analysis, cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors (Roche Life Science, Switzerland).. The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4°C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4°C); rabbit-EGFR (#4267, CST; 1:1,000 overnight, 4°C); rabbit-caspase 3 (19677-1-AP, proteintech, 1:1000 overnight, 4°C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4°C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4°C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4°C); rabbit-AR (#5153, CST, 1:1000 overnight, 4°C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4°C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4°C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4°C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4°C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-pSTAT3 (#9145, CST, 1:1,000 overnight, 4°C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4°C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4°C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4°C); mouseGAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4°C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4°C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4°C).. For immuneprecipitation, cells were treated by MG132 (10 µg/mL, Beyotime, China) for 4h before lysed in IP-RIPA buffer with protease inhibitors, and Protein A/G plus Agarose (Santa Cruz, USA) were used to pull down the proteins.

Article Title: FBXW2 Inhibits Prostate Cancer Progression and Metastasis Via Promoting EGFR Ubiquitylation and Degradation
Article Snippet: Immunoblotting (IB) and Immunoprecipitation (IP) For direct IB analysis, cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors (Roche Life Science, Switzerland).. The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4°C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4°C); rabbit-EGFR (#4267, CST; 1:1,000 overnight, 4°C); rabbit-caspase 3 (19677-1-AP, proteintech, 1:1000 overnight, 4°C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4°C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4°C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4°C); rabbit-AR (#5153, CST, 1:1000 overnight, 4°C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4°C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4°C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4°C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4°C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-pSTAT3 (#9145, CST, 1:1,000 overnight, 4°C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4°C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4°C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4°C); mouseGAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4°C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4°C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4°C).. For immuneprecipitation, cells were treated by MG132 (10 μg/mL, Beyotime, China) for 4h before lysed in IP-RIPA buffer with protease inhibitors, and Protein A/G plus Agarose (Santa Cruz, USA) were used to pull down the proteins.

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor.
Article Snippet: For direct IB analysis, cells were lysed in a Triton X-100 or RIPA buffer with phosphatase inhibitors.. The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 C); rabbit-b-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 C); mouse-b-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 C).. To immunoprecipitate endogenous proteins, whole cell extracts were pre-cleared with normal IgG-AC (Santa Cruz) followed by overnight incubation at 4 C with antibody against FBXW2, b-TrCP, FBXL3, FBXL11 or SKP2; to immunoprecipitate exogenously expressed FLAG-tagged or HA-tagged proteins, the pre-cleared cell lysates were incubated with bead-conjugated FLAG (Sigma) or with HA antibody (Roche) in a rotating incubator overnight at 4 C. The immunoprecipitates were washed with lysis buffer and assessed by IB.

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor
Article Snippet: For direct IB analysis, cells were lysed in a Triton X-100 or RIPA buffer with phosphatase inhibitors.. The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).. To immunoprecipitate endogenous proteins, whole cell extracts were pre-cleared with normal IgG-AC (Santa Cruz) followed by overnight incubation at 4 °C with antibody against FBXW2, β-TrCP, FBXL3, FBXL11 or SKP2; to immunoprecipitate exogenously expressed FLAG-tagged or HA-tagged proteins, the pre-cleared cell lysates were incubated with bead-conjugated FLAG (Sigma) or with HA antibody (Roche) in a rotating incubator overnight at 4 °C.

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