Goat anti sec23

Glomeruli were isolated by perfusion with Dynabeads^TM and were subjected to glass-glass homogenization in lysis buffer (RIPA buffer) [52 (link)]. Cells were harvested by trypsinization, and lysis was performed using RIPA buffer. The protein concentration was determined with a BCA assay (Pierce Biotechnology, Waltham, MA, USA, 23225). Equal amounts of protein were separated on SDS–PAGE gels. Then, the proteins on the gels were transferred to a polyvinyl difluoride membrane (Trans-Blot^® Transfer Pack, Bio-Rad, Hercules, CA, USA, 1704157) with a semidry blotting technique (Trans-Blot^® Turbo™ Transfer System, Bio-Rad, Hercules, CA, USA, 1704150). The membranes were blocked in 5% PBS-BSA. The following antibodies were used for Western blotting: rabbit anti-BECLIN 1 (Cell Signaling, Danvers, MA, USA, 3738S), rabbit anti-VEGF (Abcam, Cambridge, UK, 51745), anti-β-actin (Sigma, St. Louis, MI, USA, A5441), rabbit anti-LC3B (Cell Signaling, Danvers, MA, USA, 2775), rabbit anti-β-COP (Abcam, Cambridge, UK, 2899), goat anti-SEC23 (Abcam, Cambridge, UK, 99552), and rabbit anti-RAB5 (Abcam, Cambridge, UK, 218624). Densitometry was performed using LabImage ID software (Kapelan Bio-Imaging).

Rabbit antiserum against DSP portion of mouse DSPP (LF-153) was as described [60 (link)]. Other primary antibodies used for western analysis include mouse anti-Flag M2 (1:1,000, Sigma, Cat #: F1804), mouse anti-Myc (1:1,000, Cell Signaling Tech. Cat #: 2276), mouse anti-β-Actin (1:1,000, Sigma, Cat #: A1978), rabbit anti-HA (1:1,000, Cell Signaling Tech. Cat #: 3724), goat anti-hGH (1:500, Santa Cruz Biotech. Cat #: sc-10365), goat anti-SEC23 (1:200, Abcam, Cat #: AB99522), mouse anti-CALR (FMC75) (1:1,000, Calbiochem, Cat #: 208912), rabbit anti-cyclophilin B (1:1,000, Abcam, Cat #: ab16045) and custom-prepared and affinity-purified rabbit anti-S4CT (0.5 μg/ml). Secondary antibodies include IRDye 680 donkey anti-rabbit IgG, IRDye 800 donkey anti-mouse IgG, and IRDye 680 donkey anti-goat IgG (1:20,000, LI-COR Biosciences).