32-3300

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), K-562 (Lane 2), COS-7 (Lane 3), Jurkat (Lane 4), LNCaP (Lane 5) HEK-293 (Lane 6) and Caco-2 (Lane 7). The blot was probed with Anti-SKP2/p45 Mouse Monoclonal Antibody (Product # 32-3300, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjµgate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~48 kDa band corresponding to SKP2/p45 was observed across the cell lines tested. An additional band of ~46 kDa corresponds to the splice-variant of SKP2/p45. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of SKP2 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR738369_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of SKP2 was performed by loading 30 µg of HeLa wild type (Lane 1) andHeLa SKP2 KO (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with SKP2 Monoclonal Antibody (SKP2-8D9) (Product # 32-3300, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10,000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to SKP2.An uncharacterized band was observed at ~40 kDa in wild type sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of SKP2 was achieved by transfecting SH-SY5Y cells with SKP2 specific siRNAs (Silencer® select Product # s12894). Western blot analysis (Fig. a) was performed using Modified whole cell extracts (1%SDS) from the SKP2 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-SKP2 Monoclonal Antibody (SKP2-8D9) (Product # 32-3300, 2µg/ml) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this Western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to SKP2..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Validation of SMIP001 and SMIP004 . (a) Chemical structure of the two small molecule inhibitors of p27 depletions (SMIPs) identified in the screen. (b) Cell lysates from LNCaP-S14 treated with 40 muM SMIPs for 24 h were analysed by immunoblotting. Roscovitine (R) was used as positive control. The data shown is representative of two independent experiments. (c) LNCaP-S14 cells were treated with increasing concentrations of SMIPs (5-40 muM) for 18 h followed by fixation and staining for p27. The graph represents data from 24 replicate wells per concentration. (d) LNCaP-S14 cells were treated with SMIPs (5-40 muM) for 18 h and analysed by immunoblotting for the levels of p27 and p21. (e) p27, p21 and SKP2 stability in LNCaP-S14 cells treated with 40 muM SMIPs for 18 h. Cycloheximide (CHX, 100 mug/mL) was added for the indicated time points prior to preparation of cell lysates and immunoblot analysis. MG132 was used as positive control. The graphs represent the quantification of p27, p21 and SKP2 levels relative to the zero hour time point. The data is representative of three independent experiments. (f) LNCaP-S14 cells were treated with SMIP00s (40 muM) for 18 h followed by extraction of total RNA. The mRNA levels of p27, p21 and SKP2 were quantified by quantitative polymerase chain reaction as described in the Methods section. All data was normalized to GAPDH and is expressed as fold induction. The graph is representative of two independent experiments. (g) Total lysat

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Effect of MIB on p27, SKP2, and other cell cycle regulators ( A ) LNCaP cells were incubated with 10 ng/ml MIB for the indicated periods. Cell lysate was harvested and expression of the indicated proteins was assessed by immunoblotting with respective antibodies. Tubulin is shown as a loading control. SKP2 and p27 protein levels were quantitated using a demonstration copy of the Totallab software from Nonlinear Dynamics. ( B ) LNCaP cells were incubated with the indicated concentrations of MIB for 72 h, followed by preparation of total cell lysate and immunoblotting with antibodies against p27, SKP2, and tubulin. The effects of androgen on p27 and SKP2 were maximal at 1 ng/ml MIB. ( C ) LNCaP cells were maintained in the presence of 1 ng/ml MIB for 96 h after which the medium was replaced and 750 ng/ml of the antiandrogen cyproterone acetate (CA) was added. Cell lysates were prepared after the indicated times, and p27 and SKP2 levels were assessed by immunoblotting. ( D ) LNCaP cells were incubated with 1 ng/ml MIB and/or 750 ng/ml CA for 72 h and cell lysates were prepared. Expression of the indicated proteins was assessed by immunoblotting. Tubulin levels are shown as loading controls. ( E ) LNCaP cells were incubated with 1 ng/ml MIB and/or 750 ng/ml CA for 72 h and cells were fixed for flow cytometry. Cell cycle profiles and the fraction of cells in each cell cycle phase are shown. ( F ) LNCaP cells were maintained in the absence or presence of 10 ng/ml MIB for 7

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 CSN6 enhances Fbxw7's autoubiquitination and degradation (a) CSN6 increased Fbxw7alpha turnover. 293T cells were infected with lentivirus to express CSN6 shRNA or luciferase control shRNA. The cells were then transfected with Flag-Fbxw7alpha and were then treated with cycloheximide (CHX; 100 ug/ml) for the indicated times. The density of Fbxw7alpha was measured and the integrated optical density (OD) was measured. The turnover of Fbxw7alpha is indicated graphically. (b) CSN6-mediated Fbxw7alpha downregulation was proteasome-dependent. 293T cells were cotransfected with indicated plasmids and were treated with or without 50 ug/ml MG132 for 6 hours. The cell lysates were then immunoblotted with the indicated antibodies. (c) CSN6 enhanced Fbxw7alpha ubiquitination through lysine 48 linkage. 293T cells were cotransfected with Flag-Fbxw7alpha with or without GFP-CSN6 plus His-ubiquitin wild type (wt), K63R mutant, or K48R mutant. Cells were treated with 50 ug/ml MG132 for 6 hours before harvest. The ubiquitinated Fbxw7alpha proteins were pulled down using Ni-NTA-agarose beads and detected with anti-Flag antibody. (d) An F-box domain deletion mutant of Fbxw7alpha was resistant to CSN6-mediated ubiquitination. 293T cells were cotransfected with GFP-CSN6, His-ubiquitin plus Flag-Fbxw7alpha wild-type or F-box domain deletion mutant (DeltaF). Cells were treated with 50 ug/ml MG132 for 6 hours before harvest. The ubiquitinated Fbxw7alpha proteins were pulled down using Ni-