- 38-0200 - Invitrogen Antibodies PSMD14 antibody

Submitted references BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair.

Zhang S, Peng X, Li X, Liu H, Zhao B, Elkabets M, Liu Y, Wang W, Wang R, Zhong Y, Kong D
Cell death & disease 2021 May 26;12(6):546

Aggresome-Like Formation Promotes Resistance to Proteotoxicity in Cells from Long-Lived Species.

Sunchu B, Riordan RT, Yu Z, Almog I, Dimas-Munoz J, Drake AC, Perez VI
The journals of gerontology. Series A, Biological sciences and medical sciences 2020 Jul 13;75(8):1439-1447

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of (A) HeLa and (B) NIH 3T3 cell lysates using Rb anti-POH1 (C-term) (Product # 38-0200).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of Hep G2 (Lane 1), HeLa (Lane 2), A-431 (Lane 3), K-562 (Lane 4), MCF7 (Lane 5), HT-29 (Lane 6), HCT 116 (Lane 7) and Caco-2 (Lane 8). The blot was probed with Rabbit Anti-PSMD14 Polyclonal Antibody (Product # 38-0200, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 35 kDa band corresponding to PSMD14 was observed across the cell lines tested. 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 following blocking with 5 % skimmed milk. 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: Knockdown of PSMD14 was achieved by transfecting HeLa cells with PSMD14 specific validated siRNAs (Silencer® select Product # s19921, Product # s19920). Western blot analysis (Fig. 1) was performed using whole cell extracts from the PSMD14 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-PSMD14 Polyclonal Antibody (Product # 38-0200, 1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. 2). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PSMD14.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of PSMD14 was performed using 70% confluent log phase HepG2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with PSMD14 Rabbit Polyclonal Antibody (Product # 38-0200) at 5µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 Effects of BKM120 and rucaparib as single agents or in combination on HR repair. A U251 and U87MG cells were transfected with DR-GFP plasmid and pCBASceI plasmid using Lipofectamine 3000. Cells were treated with BKM120 or/and rucaparib for 72 h and then collected and resuspended in ice-cold PBS. GFP intensity was analyzed by flow cytometry. B The percentage of GFP-positive cells represents HR repair efficiency. C , D U251 and U87MG cells were treated with BKM120 and/or rucaparib for 48 h. C Cells were collected for western blotting detection of DNA damage- and HR repair-related proteins, including BRCA1, BRCA2, RAD51, RRM2, p-ATR, p-CHK1, p-AKT, ATR, CHK1, and PAR. D Quantitative reverse transcription PCR analysis of BRCA1, BRCA2, and RAD51 expression in two cancer cell lines. Gene expression was normalized to 18 s rRNA. E , F Cells were treated with BKM120 or/and Rucaparib for 12/24 h. Representative images of immunofluorescence staining of RAD51 and gamma-H2AX in GBM cells Cell nuclei were stained with DAPI. Scale bar: 20 mum. The percentage of cells with RAD51/gamma-H2AX foci was shown in ( F ). All data are presented as mean +- SD ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001.

38-0200

Antibody data