Western blot analysis was performed on whole-cell extracts (30 µg lysate) of K-562 (Lane 1), Hep G2 (Lane 2), HeLa (Lane 3), Jurkat (Lane 4), HEK-293 (Lane 5), U-2 OS (Lane 6), and A549 (Lane 7). The blots were probed with Rabbit Anti-PSMD7 Polyclonal Antibody (Product # PA1-1963, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 40 kDa band corresponding to PSMD7 was observed across the cell lines tested. In addition, a ~50 kDa band was observed, which could possibly represent the 26s Proteasome Subunit S5B. 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).
Immunofluorescence analysis of PSMD7 was performed using 70% confluent log phase Hep G2 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 PSMD7 Rabbit Polyclonal Antibody (Product # PA1-1963) at 2 µg/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) at 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytosolic localization. Panel e shows the control without primary antibody. The images were captured at 60X magnification.
Supplier Page from Thermo Fisher Scientific for PSMD7 Antibody