Thrombospondin

For the validation of protein expression changes by immunoblotting, 20 μg protein extract was separated on 8% or 12% SDS polyacrylamide gels according to Laemmli [43 (link)]. Proteins were transferred to nitrocellulose membranes (GE Healthcare) using a semidry blotting system at 100 mA for 90 min. Membranes were saturated for one hour with 5% advance blocking reagent (GE Healthcare) in TBS (50 mM Tris.HCl, pH 7.6 and 150 mM NaCl) containing 0.1% Tween 20 (TBS/T). Blots were incubated overnight at +4°C with antibodies against either cMYC (Santa Cruz Biotechnology, Inc.) or thrombospondin (Abcam).
After washing three times in Tris-buffered saline/Tween 20 TBS/T, blots were incubated for one hour at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-goat secondary antibody (Santa Cruz Biotechnology) in blocking buffer (TBS/T with 5% w/v advance blocking reagent). Immunodetection was performed either with ECL advance Western blotting detection kit (GE Healthcare) following standard procedures. The protein bands were quantified using ImageQuant 5.2 software (GE Healthcare) by integration of all pixel values in the band area after background correction and normalised to the loading control, RAD50 (GeneTex, Taiwan) or actin (Santa Cruz Biotechnology, Inc., US).

Platelet morphology in the Spanish index was assessed by light and immunofluorescence microscopy on blood smears as reported [46 (link),47 (link)]. Particularly, the expression of three α-granule markers (i.e., thrombospondin, von Willebrand factor (vWF), and P-selectin), three dense granule markers (i.e., markers LAMP-1, LAMP-2, and CD63), and the cytoskeletal protein ß-tubulin was determined. The following primary antibodies were used: anti-thrombospondin (ab85762, Abcam, Cambridge, UK); anti-P-selectin (555522, BD Biosciences, San Jose, CA, USA); anti-vWF (A0082, Dako, Waldbronn, Germany); anti-LAMP-1 (sc18821, Santa Cruz Biotechnology, Heidelberg, Germany); anti-LAMP-2 (sc18822, Santa Cruz, CA, USA); anti-CD63 (558019, BD Biosciences); anti-β1-tubulin (T4026, Merck Life Science, Darmstadt, Germany). As secondary antibodies, Alexa Fluor 568 (A11011, Invitrogen, Thermo Fisher Scientific) and Alexa Fluor 488 (A11001, Invitrogen, Thermo Fisher Scientific, Dreieich, Germany) were used. Each marker was eventually assessed by standard immunofluorescence microscopy using the Olympus BX40 microscope system (Olympus, Hamburg, Germany). Blood smears from healthy controls were stained and analyzed in parallel.

The following antibodies were used: V5 was used to visualize GFP-Apex and S1R-Apex (ThermoFisher; 1:10,000 for western, 1:1,000 for immunofluorescence); Bip (Cell Signaling; 1:1,000 for western); PDI (Cell Signaling; 1:1,000 for western); Calnexin (Cell Signaling; 1:1,000 for western); Sec61alpha (Santa Cruz; 1:100 for western); Sigma1 Receptor (1:500 for western) (Ola et al., 2002 (link)); Lman1 (Abcam; 1:1,000 for western); PCSK9 (Abcam; 1:3,000 for western, 1:300 for immunofluorescence); LDLR (Novus biologicals; 1:1,000 for western, 1:100 for immunofluorescence); Thrombospondin (Abcam; 1:250 for western); Gapdh (Santa Cruz; 1:2000 for western).