MA5-12717

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A549 (Lane 1) and HEL 92.1.7 (Lane 2). The blots were probed with Anti-Cyclin D3 Mouse Monoclonal Antibody (Product # MA5-12717, 1:500-1:2000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A 32 kDa band corresponding to Cyclin D3 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then onto a 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)
Main image
Experimental details: Knockdown of Cyclin D3 was achieved by transfecting A549 cells with Cyclin D3 specific validated siRNAs (Silencer® select Product # s2522). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the Cyclin D3 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Cyclin D3 Monoclonal Antibody (Product # MA5-12717, 1 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 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 Cyclin D3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on modified whole cell extracts (1% SDS) of A549 (Lane 1), K562 (Lane 2) and PC-12 (Lane 3). The blot was probed with Anti-Cyclin D3 antibody (Product # MA5-12717, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 32 kDa band corresponding to Cyclin D3 was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of CCND3 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042) (Assay ID CRISPR796863_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of CCND3 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2), HeLa CCND3 KO (Lane 3) whole cell extracts. The blot was probed with Anti-Cyclin D3 Monoclonal Antibody (DCS-22)(Product # MA5-12717) using 1 µg/mL dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to CCND3.