Rabbit α galectin 1

Following fixation with 4% paraformaldehyde for 10 min, cells were washed 3 times for 5 min with 0.1 M phosphate buffer (0.1 M Na₂HPO₄ × 2H₂O, 0.1 M NaH₂PO₄ × H₂O, pH 7.4) and blocked with 3% bovine serum albumin and 0.1% Triton X-100 in 0.1 M phosphate buffer for 30 min. After incubation at 4 °C overnight with 1:50 rabbit α-galectin-1 (Abcam, Cambridge, UK), 1:100 mouse α-smooth muscle-α-actin (Santa Cruz, Dallas, TX, USA), 1:100 mouse α-N-cadherin (Thermo Fisher), 1:100 mouse α-integrin-β1 (Merck), 1:100 mouse α-cytokeratin-8 (Merck), 1:100 phalloidin Alexa Fluor 555 (Thermo Fisher) in 0.3% bovine serum albumin, and 0.01% Triton X-100 in 0.1 M phosphate buffer, specimens were washed again 3 times for 10 min each with 0.1 M phosphate buffer and incubated with 1:1000 goat anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 488 (both from Thermo Fisher) in 1:10 diluted blocking solution for 1 h at room temperature. After nuclear staining with Hoechst 33342 (Thermo Fisher), specimens were washed again 3 times with 0.1 M phosphate buffer and mounted with the ProLong glass antifade mounting medium (Thermo Fisher). Immunofluorescence staining was analyzed by an Axio Observer 7 fluorescence microscope with an Apotome 2 module (Zeiss) and documented using the ZEN software Blue edition 3.0 (Zeiss).

ARPE-19 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Complete; Merck). Up to 15 µg of the total protein per sample was loaded onto a 12% SDS-PAGE and transferred onto a PVDF membrane (Roche, Mannheim, Germany) by semidry blotting following electrophoresis. After blocking with 3% skimmed milk powder in 1× PBST, membranes were incubated with 1:1000 rabbit α-galectin-1 (Abcam, Cambridge, England) in 1× PBS with 0.3% skimmed milk powder and 0.1% Tween 20 at 4 °C overnight. Following 3 washes with 1× PBS for 10 min each, the membranes were hybridized with 1:1000 alkaline phosphatase (AP)-conjugated goat α-rabbit antibody (DIANOVA, Hamburg, Germany) in 1× PBS with 0.3% skimmed milk powder and 0.1% Tween 20. As loading control, blots were incubated with 1:5000 goat α-tubulin antibodies (R&D System, Minneapolis, MN, USA) in 1× PBS with 0.3% skimmed milk powder and 0.1% Tween 20 overnight, followed by an additional hybridization with 1:1000 AP-conjugated donkey α-goat antibodies (Dianova, Hamburg, Germany) in 1× PBS with 0.3% skimmed milk powder and 0.1% Tween 20 for 1 h. For visualization, membranes were incubated with CDP-Star substrate in accordance with the manufacturer’s recommendations (CDP-Star, Thermo Fisher) and documented with an iBrightCL1000 Imaging System (Thermo Fischer).