Ab1543p

Protein levels were quantified using an automated capillary-based size sorting system (O'Neill et al., 2006 (link)), “WES” from ProteinSimple. All procedures were performed with manufacturers reagents according to the user manual. Briefly, 8 μL of cell lysate was mixed with 2 μL of 5× fluorescent master mix and heated at 95°C for 5 min. The samples, blocking reagent, wash buffer, primary antibodies, secondary antibodies, and chemiluminescent substrate were dispensed into designated wells in the manufacturer provided microplate. Following plate loading the separation and immunodetection was performed automatically using default settings. The data was analyzed with inbuilt Compass software (Proteinsimple). Primary antibodies used were α-synapsin 1 (rabbit, Millipore, AB1543P, 1:7500), α-phosphoserine 603 synapsin 1 (rabbit, Cell Signaling, 2311, 1:2500), and α-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, rabbit, Cell Signaling, 2118S).

Cultures were scraped from individual coverslips at DIV21 in 100 μl of LDS loading buffer (Life Technologies). 15 μl of lysate was resolved using SDS-PAGE on a NuPage 4–12% Bis-Tris gel (Life technologies) and transferred onto a PVDF membrane (Millipore) at 25 V for 100 min at room temperature. Membranes were incubated overnight in 2% BSA/TBST with the following primary antibodies; α-Endophilin A (EndoA, mouse, Thermo Scientific, WH0006456M1, 1:1000), α-vesicle associated membrane protein 1 (VAMP1, rabbit, Synaptic Systems, 104 002, 1:2500), α-vesicle associated membrane protein 2 (VAMP2, rabbit, Synaptic Systems, 104 202, 1:2500), α-synaptojanin 1 (SynJ1, rabbit, Synaptic Systems, 145 003, 1:1000), α-dynamin 1 (DNM1, rabbit, Thermo Scientific, PA1-660, 1:1000), α-synapsin 1 (rabbit, Millipore, AB1543P, 1:5000), α-phophoserine 9 synapsin 1 (rabbit, Thermo Scientific, PA1-4604, 1:2500), α-phosphoserine 603 synapsin 1 (rabbit, Cell Signaling, 2311, 1:2500). Secondary antibodies (HRP conjugated α-mouse and α-rabbit, Santa Cruz Biotechnology) were used at 1:5000. BIO-RAD ChemiDoc™ MP Imaging System was used to detect the signal, which was quantified using inbuilt Image Lab software (Bio-Rad).

Protein expression analysis was performed using a Jess automated system (ProteinSimple, Santa Clara, CA, USA) according to the ProteinSimple user manual. In brief, tissue lysate samples were mixed into a master mix (ProteinSimple) containing sample buffer and DTT to the final concentration and then heated at 95 °C for 5 min. The samples, blocking reagent, primary antibodies, HRP- or NIR-conjugated secondary antibodies, luminol–peroxide mix, and wash buffer were added to designated wells in a 384-well plate. After plate loading, the separation electrophoresis and immunodetection steps proceeded in the capillary system and were fully automated in the Jess automated system. The digital images were analyzed with Compass software (ProteinSimple), and the quantified data of the detected proteins were reported as molecular weight and signal/peak intensity. The target protein antibodies were BDNF (1:25; NB100-98,682, NOVUS), BCL2 (1:25; SC-7382, NOVUS), BAX (1:50; D2E11, Cell Signaling), CASP3 (1:25; NB100-23,708, NOVUS), BRN3A (1:50; MAB1585, Millipore), ISLET1 (1:50; NBP2-14,999, NOVUS), SYNAPSIN1 (1:25; AB1543P, Millipore), SYNAPTOTAGMIN (1:25; MAB5200, Millipore), and GAPDH (1:100; 60,004-1-Ig, Proteintech). For quantitative data analysis, all of the target signals were normalized against GAPDH as a loading control.