Alexa fluor 488 conjugated goat anti mouse igg a 11001

An aliquot of washed erythrocytes from above was fixed in 5% formaldehyde (Sigma-Aldrich, Missouri, USA) on the day of venepuncture and CR1 copy number was determined within 8 weeks as previously described in detail^{65 (link)}. Briefly, this method uses flow cytometry to quantify CR1 copy number on fixed erythrocytes using a standard curve derived from donors with known CR1 copy numbers that have been fixed at the same time as the samples. Erythrocytes were stained with 0.5 μg/ml J3D3 anti-CR1 monoclonal antibody (GTX44217, GeneTex, California, USA), followed by 5 μg/ml of Alexa Fluor 488-conjugated goat anti-mouse IgG (A-11001, ThermoFisher Scientific, Paisley, UK) and the mean fluorescence intensity determined on a FC500 flow cytometer (Beckman-Coulter Inc.). Examples of the gating strategy and staining of low, intermediate and high CR1 standard donors are shown in Figure S6.

Recombinant human PCSK9 (rhPCSK9, gl-10916) was purchased from Genlocus (Chengdu, China). βE2 (E2758-1G) was purchased from Sigma-Aldrich (Shanghai, China). The GPER antagonist G15 (1161002-05-6) was purchased from Cayman Chemical (Michigan, USA). The PLC inhibitor U73122 (U6756-5 MG) was purchased from Sigma-Aldrich (San Francisco, USA). Anti-LDLR antibody (sc-373830) was ordered from Santa Cruz (Dallas, USA). Anti-phospho-PLCγ (Tyr783) polyclonal antibody (2821) and anti-PLCγ rabbit antibody (2822) were purchased from Cell Signaling (Hongkong, China). Anti-clathrin rabbit polyclonal antibody (ab59710) was purchased from Abcam (Shanghai, China). The BODIPY^TM FL LDL (L3483), Alexa Fluor^® 488 protein labeling kit (A10235), Alexa Fluor^® 488-conjugated goat anti-mouse IgG (A11001) and Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (A11008) were all purchased from Thermo Fisher (Shanghai, China). A cAMP parameter assay kit (KGE002B) was purchased from R&D (Minneapolis, USA). The PrimeScript™ II 1st Strand cDNA synthesis kit (6210A) and TB GreenTM Premix Ex Taq II (RR420B) were purchased from TaKaRa (Dalian, China).