Tran blot turbo transfer system

Unaffected control (n = 3), affected (n = 3), and ABE-treated (n = 3) HDF cell pellets were lysed in CelLytic M (Sigma-Aldrich), and protein concentration was estimated by BCA assay (ThermoFisher Scientific). A total of 8 μg protein was resolved in 4%–15% mini-PROTEAN TGX Stain-Free gels (Bio-Rad) and transferred to PVDF membranes using the Tran-Blot Turbo Transfer System (Bio-Rad) Membranes were then blocked in 2% casein for 1 h before overnight incubation with one of the following primary antibodies: LAMP1 mouse monoclonal antibody (1:1,000, Abcam; RRID: AB_470708) or CD63 mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology; RRID: AB_627877). Loading controls for LAMP1 and CD63 are GAPDH (1:2,500, Novus Biologicals; RRID: AB_10002458) and Vinculin (1:1,000, Cell Signaling Technology; RRID: AB_2728768) antibodies, respectively. Secondary antibody (anti-mouse-HRP Bio-Rad; RRID: AB_11125547; anti-rabbit-HRP, Bio-Rad Anti-Rabbit-HRP; RRID: AB_11125142 all 1:3,000) were applied to the membrane at room temperature for 45 min. Clarity Western ECL Substrate (Bio-Rad 170-5060) was applied and ECL signals were captured by Bio-Rad ChemiDoc Imaging System and quantified with FIJI software.³⁴ (link)

Equal numbers of cultured neurons underwent treatment, were lysed in equal volumes of lysis buffer (1× NuPAGE lithium dodecyl sulfate sample buffer and 1× NuPAGE Sample Reducing Agent), were denatured at 95 °C for 10 min, and were loaded per gel lane. Protein samples were electrophoresed on NuPAGE Novex 4 to 12% Bis-Tris Midi protein gels in 1× NuPAGE MOPS or MES SDS running buffer at 200V for 1 h at RT. Gels were transferred to nitrocellulose membranes with Tran-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with Odyssey blocking buffer (Li-Cor 927–40000) at RT for at least 1 h, incubated with primary antibodies overnight at 4 °C followed by 4× washes with TRIS-buffered saline with Tween at RT, and then incubated with secondary antibodies at RT for 1 h, followed by 4× washes with TRIS-buffered saline with Tween at RT. Li-Cor odyssey system was used for Western blot detection and quantification. The band intensities of proteins of interest were quantified using Image Studio Lite software (Li-Cor; https://www.licor.com/bio/image-studio-lite/) and normalized to those of loading control protein (e.g. Tuj1) per each lane. To quantify the calpain activity (Fig. 6), the signal intensities of full-length and cleaved substrates were separately quantified. Images of the full uncropped Western blot images can be found in the online Supporting Information.