Ab30682

To verify the antibody specificity, transient expression of the targeted proteins (full-length human SREBP2: Addgene cat #26807; full-length human HMGCS1: in-house; full-length human HMGCR: Addgene cat #86085; GFP-tagged full-length human LDLR: Addgene cat #98184), or gene knockdown using the SMARTpool siRNAs mentioned above. In brief, MO3.13 cells were seeded in the 6-well plates 1 d before transfection. The next day, the cells were transfected with the plasmids or the SMARTpool siRNAs by the use of Lipofectamine 2000. The cells were placed in a 37°C incubator for 72 h, with the daily medium changing. The cells were then harvested and lysed in RIPA buffer. To verify the antibody specificity and localization, gene was knockdown using the SMARTpool siRNAs mentioned above. In brief, HeLa cells were seeded on 12-mm glass coverslips. The next day, the cells were transfected with SMARTpool siRNAs by the use of DharmaFECT 1. The cells were placed in a 37°C incubator for 48 h, with a change of media after 24 h. The cells were then fixed with 4% PFA/4% sucrose. IF was performed as mentioned above with the following primary antibodies: SREBP2 (Abcam; ab30682; 1:200), LDLR (Proteintech; 10785–1-AP; 1:500), HMGCS1 (Proteintech; 17643–1-AP; 1:1,000) and HMGCR (Proteintech; 13533–1-AP; 1:500).