Primescript 2 1st strand cdna synthesis kit 6210a

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).

Total RNA was extracted from hyphae using the RNAiso Plus 9108 (TaKaRa, Japan) according to the manufacturer’s instructions. The isolated RNA (1.0 μg) was used to synthesize cDNA via the PrimeScriptII 1st Strand cDNA Synthesis Kit 6210A (TaKaRa, Japan). The quantitative real-time PCR (qRT-PCR) analysis was performed with a LightCycler® 480 II Real-Time PCR Detection System (Roche, Basel, Swiss) using SYBR Premix Ex Taq RR820A (TaKaRa, Japan) and β-tubulin gene as the internal control. Primers used in this study are listed in Table S2. All the reactions were performed with three biological and technical replicates independently.