Distribution of FAIML-immunoreactive cells in the hippocampus of mice brain (3). Immunohistochemistry of P10 hippocampus sample. Sections of 100 uM were incubated overnight with the antibody. This primary antibody was visualized by sequential incubation with biotinylated secondary antibodies (1:200; Vector Laboratories, Burlingame, CA) and the streptavidine-peroxidase complex (1:400; Amersham Biosciences, Pittsburgh, PA). The peroxidase reaction was developed with diaminobenzidine and H2O2 . Some hippocampal interneurons scattered in all hippocampal layers show a strong immunostained signal (arrows). So, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare; sm, stratum moleculare.
Immunofluorescence localization (arrows) of FAIML proteins in cortical layer V at P5 (3). After blocking, sections were incubated overnight with antibody (1:600), and was visualized using secondary Alexa Fluorconjugated antibodies. Sections were counterstained with Bisbenzimide, mounted onto slides, and viewed under confocal microscopy.
Immunoblot analysis of the FAIML expression in human and mouse hematopoietic/immune cells (3). Cortical neurons, neuroblastoma SH-SY5Y, and transfected PC12 extracts were used as a positive control of the FAIML and total FAIM expression. 20 ug of total protein were resolved by SDS-PAGE and blotted with anti- FAIMS (top panel) and FAIML (middle panel). Napthol blue staining was used as a loading control (bottom panel).
The Western Blot signal, coming from endogenous FAIM-L, is reduced in extracts from cultured cells with FAIM-L gene silencing driven by shRNA interference (3). Scheme of FAIML- and FAIMS-targeted RNAi design. Positions for the different RNAi are indicated, R1 is sequence specific for FAIML, and R2 is in the common sequence and specifically silences the FAIMS (top panel). PC12 cells were infected with R1, R2, or RS (scrambled) for 72 h, and the effects on endogenous FAIMS/L expression were analyzed by Western blot with anti-FAIM antibodies (bottom panel). α-Tubulin was used as a loading control.