Anti cd109

Cell lysates (containing 20 μg total protein) were analyzed by western blot with one of the following antibodies: mouse monoclonal anti-CD109 (BD Biosciences; 556039), anti-fibronectin (BD Biosciences; 610078), anti-N Cadherin (Abcam; ab18203), anti-E-Cadherin (Abcam; ab216783), anti-p44/42 Erk1/2 (Cell Signaling, Danvers, MA, USA; mAb #4695), anti-STAT3 (Abcam; ab5073), Anti-phospho-Stat3 (Tyr705) (Cell Signaling, Danvers, MA, USA; #9131S), Anti-Akt (Cell Signaling, Danvers, MA, USA; mAb #4685), Anti-phospho-Akt (Ser473) (Cell Signaling, Danvers, MA, USA; mAb #4060), anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-47778). Briefly, western blot analysis involved separating cell extracts by SDS-polyacrylamide gel electrophoresis (PAGE) and transferring to polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin/TBST blocking buffer at room temperature for 30 min and then incubated overnight at 4 °C with specific primary antibodies. The membranes were washed with TBST wash buffer followed by incubation with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Bands were detected with an enhanced chemiluminescence (ECL) system (MilliporeSigma, Burlington, MA, USA; WBKLS0500).

HEK293 (ATCC CRL-1573) cells and their derivative cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂. PGAP1-KO^{69 (link)} and PGAP2-KO^{70 (link)} cells have been constructed previously. KO cell lines used in this study are listed in Supplementary Table 1. Mouse monoclonal anti-CD55 (clone IA10)^{50 (link)}, anti-CD59 (clone 5H8)^{50 (link)}, anti-CD230 (14-9230-82; Thermo Fisher Scientific), anti-CD109 (556039; BD Biosciences), anti-GAPDH (60004-1-Ig, clone 1E6D9; Proteintech), anti-FLAG (F3165; M2; Sigma-Aldrich) and rabbit monoclonal anti-HA (3724S; Cell Signaling Technology) were used as primary antibodies. F(ab’)2-goat anti-mouse IgG, PE (12-4010-82; Thermo Fisher Scientific) and F(ab’)2-donkey anti-rabbit IgG, PE (12-4739-81; Thermo Fisher Scientific), and Goat Anti-Mouse IgG, HRP (HS201-1; TransGen Biotech) were used as the secondary antibodies. For flow cytometric analysis, antibodies were used at 10 µg/ml. For western blotting, the primary antibodies and the secondary antibodies were used at 1 and 0.2 µg/ml, respectively. PI-PLC (Thermo Fisher Scientific) and proaerolysin (produced and purified in the laboratory) were used for treatments. Cell Counting Kit-8 (CCK-8, MedChemExpress) was used to detect cell viability.