PMC

TBS homogenates of brains from wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mice were electrophoresed on 10% tris-glycine polyacrylamide gels and western blotted with antibodies recognizing either FLAPLP2 (D2-II, A) or the extreme C-terminus of APLP2 (W2CT, C). Western blotting for GAPDH was included to check for equal protein loading (D). Lysates of cell lines over-expressing human wild type APLP2₇₅₁ (+), are included as positive control and TBS homogenates of brains from APLP2 knock-out mice (−) are included as negative control. APLP2s bands (indicated by arrows, A) were quantitated by densitometry and values normalized versus the WT control are presented as averages ± standard error of duplicate measurements of three animals of each genotype (B).

TBST homogenates of wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mouse brains were electrophoresed on 10–20% tris-tricine polyacrylamide gels and western blotted with the anti-APLP2 C-terminus specific antibody W2CT (A, C). Lysates of a cell line over-expressing human wild type APLP2₇₅₁ (+) are included as positive controls; while TBST homogenates of hemibrains of APLP2 knock-out mice (−) are included as negative controls. The FL and CTFs species identified (indicated by arrows in A and C respectively) were quantified by densitometry and normalized versus the WT control. Results are presented as averages ± standard error of duplicate measurements of three animals for each condition (FLAPLP2: B; APLP2 CTFs: D).

Microsomes prepared from BACE1 knock-out (KO) or wild type (WT) mouse brains were incubated at 37°C for 2 hours to allow de novo in vitro ICD production (A-C). ICDs were detected by western blot using specific antibodies for APP (C8 antibody, A), APLP1 (W1CT antibody, B) and APLP2 (W2CT antibody, C). Western blots shown in A-C are representative of three different experiments. Generation of ICDs was conducted either in presence (+) or absence (−) of protease inhibitors and insulin (PI mix). Endogenous ICDs were immunoprecipitated from mouse brains with C8, W1CT or W2CT, and immunoprecipitates analysed by western blotting with the same antibodies (D, E, F). Western blots shown in D-F are representative of two different experiments. For comparison, in vitro generated ICDs were electrophoresed alongside endogenous ICDs (D-F).

TBST homogenates of wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mouse brains were electrophoresed on 10–20% tris-tricine polyacrylamide gels and western blotted with the anti-APLP1 C-terminus specific antibody W1CT (A, C). Lysates of a cell line over-expressing human wild type APLP1₆₅₀ (+) are included as a positive control; while TBST homogenates of brains from APLP1 knock-out mice (−) are included as a negative control. The FL and CTFs species identified (indicated by arrows in A and C respectively) were quantified by densitometry and normalized versus the WT control and results are presented as averages ± standard error of duplicate measurements of three animals for each condition (FLAPLP1: B; APLP1 CTFs: D).

TBS homogenates of brains from wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mice were electrophoresed on 10% tris-glycine polyacrylamide gels and western blotted with antibodies recognizing the N-terminus (W1NT, A) and C-terminus (W1CT, C) of APLP1. Western blotting for GAPDH was included to check for equal protein loading (E). Lysates of a cell line over-expressing human APLP1₆₅₀ (+), are included as a positive control and TBS homogenates of brains from APLP1 knock-out mice (−) are included as a negative control. FLAPLP1 and APLP1s bands detected by W1NT are indicated by arrows in A. APLP1s and of FLAPLP1 levels (B and D respectively) were quantitated by densitometry and values normalized relative to WT control are presented as averages ± standard error of duplicate measurements of three animals of each genotype.

TBST homogenates of wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mouse brains were electrophoresed on 10–20% tris-tricine polyacrylamide gels and western blotted with the anti-APP C-terminus specific antibody C8 (A, C). Lysates of a cell line over-expressing human wild type APP₆₉₅ (+) are included as a positive control; while TBST homogenates of brains from APP knock-out mice (−) are included as a negative control. The asterisk in C indicates a specific band detected in certain WT and Tg samples. FL and CTF bands (indicated by arrows in B and D respectively) were quantified by densitometry and normalized versus the WT control. Results are presented as averages ± standard error of duplicate measurements of three animals for each condition.

TBS homogenates of brains from wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mice were electrophoresed on 10% tris-glycine polyacrylamide gels and western blotted with a panel of antibodies which allow detection of total APPs (22C11, A), APPsα (anti-Aβ rodent, C) and full-length and C-terminal fragments of APP (C8, E). Western blotting for GAPDH was included to check for equal protein loading (F). Lysates of a cell line over-expressing human wild type APP₆₉₅ (+), were included as a positive control and TBS homogenates of brains from APP knock-out mice (−) were included as a negative control. The levels of total APPs and of APPsα (B and D respectively) were quantitated by densitometry and values normalized versus WT control are presented as averages ± standard error of duplicate measurements of three animals of each genotype.