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. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: Apoptosis. 2021 Jan 1;26(1-2):71–82. doi: 10.1007/s10495-020-01647-9

Figure 1. Manipulation of LRG1 expression in MCF-7 cells.

Figure 1

(a) Western blot analysis of LRG1 expression in primary human mammary epithelial cells (HMEC) and in several breast cancer cell lines shows lower molecular weights compared to that of serum LRG1.

(b) Transfection of MCF-7 cells with lrg1 resulted in increased protein expression relative to actin shown by Western blotting.

(c) N-glycosidase (PNGase F) treatment of proteins extracted from MCF-7 cells resulted in a decrease in intensity of the 50 kDa band indicating that it is a fully glycosylated form of LRG1.

(d) RT-PCR analysis confirmed increased LRG1 mRNA expression in transfected cells as detected by ethidium bromide staining and decreased expression in a clone transfected with LRG1 shRNA. In the absence of reverse transcriptase (RT) there was little amplification indicating near absence of genomic DNA. Samples in the right panel were amplified through two additional cycles compared to the samples in the left panel.

(e) Western blotting confirmed decreased expression of LRG1 in the clone transfected with LRG1 shRNA and partial knock down in MCF-7 cells infected with a recombinant lentivirus encoding an LRG1 shRNA distinct from that used for transfection.

Transfection of cells with an empty vector or one containing a nonsense shRNA insert (not shown) had no effect on LRG1 expression.