Phospho-LRRK2 (S935) Recombinant Rabbit Monoclonal Antibody [JE42-11]

☑ Cell treatment (CT)

Western blot analysis of Phospho-LRRK2 (S935) on different lysates with Rabbit anti-Phospho-LRRK2 (S935) antibody (HA722315) at 1/1,000 dilution.

Lane 1: MEF cell lysate (20 µg/Lane)
Lane 2: MEF treated with 1μM okadaic acid for 1 hour cell lysate (20 µg/Lane)
Lane 3: MEF cell lysate (20 µg/Lane)
Lane 4: MEF treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)
Lane 5: NIH/3T3 cell lysate (10 µg/Lane)
Lane 6: MEF treated with 100nM Calyculin A for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour (20 µg/Lane)

Predicted band size: 286 kDa
Observed band size: 286 kDa

Exposure time: 3 minutes; ECL: K1801;
3-8% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722315) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunocytochemistry analysis of MEF cells treated with 100nM Calyculin A for 30 minutes labeling Phospho-LRRK2 (S935) with Rabbit anti-Phospho-LRRK2 (S935) antibody (HA722315) at 1/1,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-LRRK2 (S935) antibody (HA722315) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

☑ Cell treatment (CT)

Flow cytometric analysis of MEF cells (left) and MEF cells treated with 100nM Calyculin A for 30 minutes (right) labeling Phospho-LRRK2 (S935).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722315, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).