Goat anti rabbit igg hrp conjugate

Samples were denatured in Bolt LDS sample buffer and reducing agent (Thermo Fischer Scientific, B0007 and B0009) at 70 °C for 10 min. Gel electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, 4561085). Proteins were transferred to nitrocellulose membranes. After blocking in 5% milk in Tris-buffered saline (TBS), the membranes were incubated with primary antibodies (1% milk in 0.05% Tween-20 TBS, TBST) overnight at 4 °C. After washing in TBST, membranes were incubated with secondary antibodies in 1% BSA in TBST, for 1 h at room temperature. Blots were revealed using Western Lightning Plus ECL (PerkinElmer, NEL103001EA).
The following primary antibodies were used: rabbit anti-HA (Merck, H6908, 1:1,000), chicken anti-GFP (Aves Biolabs, GFP-1020, 1:10,000), rat anti-laminin-111 (R&D systems, MAB4656, 1:250, monoclonal reactivity towards LAMA1/B1). The following secondary antibodies were used: goat anti-rabbit IgG HRP conjugate (Promega, W401B, 1:5,000), goat anti-chicken IgY HRP conjugate (Thermo Fischer Scientific, A16054, 1:40,000) and rabbit anti-rat IgG HRP conjugate (Merck, A9542, 1:5,000). Uncropped blots are provided in Supplementary Fig. 1.

Cell lines (0.1 × 10⁷ cells) were seeded in a 6-well plate (Nunc) and incubated with rfhSP-D (20 µg/ml), together with an untreated control, in a serum-free DMEM-F12 medium. The cells were lysed within the wells using treatment buffer (50 mM Tris-HCl pH 6.8, 2% v/v β-mercaptoethanol, 2% v/v SDS, 0.1% w/v bromophenol blue, and 10% v/v glycerol) and transferred to pre-cooled microcentrifuge tubes followed by sonication for 15 s. The samples were heated at 100°C for 10 min and subjected to SDS-PAGE (12% w/v) for 90 min at 120 V. The SDS-PAGE separated proteins were then electrophoretically transferred onto a nitrocellulose membrane (Thermo Fisher) using an iBLOT (Thermo Fisher). The membrane was then blocked using 5% w/v dried milk powder (Sigma) in 100 ml PBS for 2 h on a rotatory shaker at room temperature. The membrane was incubated with rabbit anti-human caspase primary antibodies (anti-cleaved caspase 3; anti-cleaved caspase 8; Cell Signaling) at 4°C overnight, followed by incubation with secondary Goat anti-rabbit IgG HRP-conjugate (1:1,000; Promega) for 1 h at room temperature. The membrane was washed with PBST (PBS + 0.05% Tween 20) three times, 10 min each time. The color was developed using 3,3′-diaminobenzidine substrate kit (Thermo Fisher).