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. 2021 Dec 21;23(1):58. doi: 10.3390/ijms23010058

Figure 3.

Figure 3

NONHSAT013026.2/Linc013026-68AA is translated into a 68 amino acid long micropeptide. (A) In vitro transcription/translation assay of Linc013026-68AA, proteins are labeled with [35S]methionine. Arrow indicated the translated peptide. (B,C) GFP- and Myc-tagged Linc013026-68AA (68AA) and pEGFP-N1 (GFP) or pcDNA3.1 MycHis (Vector) vector was transfected in HeLa cells, and GFP- and Myc-specific immunoblot were performed. (D) 68AA-Myc was overexpressed in HeLa cells. Cell extracts were incubated with GST and GST-68AA and then Myc and GST specific immunoblots were performed. (E) 68AA-Myc overexpressing cell extracts were pre-treated with 1% and 2% SDS and then Myc and ACTIN specific immunoblots were performed. (F) 68AA-Myc was overexpressed in HeLa cells. Forty microliters of cell extracts were treated with 2000 U Lambda Protein Phosphatase (Lambda PP) for 45 min at 30 °C and subsequently supplied for Linc013026-68AA specific immunoblot. (G) Amino acid sequence of Linc013026-68AA was depicted. Numbers represent amino acid number. Amino acid sequences of peptide I (orange) and peptide II (blue) were used to generate rabbit antibodies. (H) Non-transfected-, pEGFP-N1 (GFP) and GFP-tagged Linc013026-68AA (68AA-GFP) HeLa cell lysates were applied for GFP-, anti-peptide I and anti-peptide II or anti-peptide II + peptide II immunoblot. ACTIN was used as loading control. (I) Myc-tagged Linc013026-68AA was expressed in HeLa and stained with anti-Myc, anti-peptide I and anti-peptide II specific antibodies and visualized by the immunofluorescent (IF) technique. (J) Myc-tagged Linc013026-68AA (68AA-Myc) and pcDNA3.1 MycHis (Vector) vector were transfected in HeLa cells and immunohistochemically stained with rabbit antibody against Linc013026-68AA-peptide I and peptide II and hematoxylin. (K) HepG2 cells were transfected with siCr and siLinc013026-68AA for three days. Total RNAs were isolated and supplied for Linc013026 or Gapdh-specific qRT-PCR. The expression of Linc013026 was normalized by Gapdh. Three independent experiments were performed. Numbers are mean ± standard deviation (SD). (L) A sister culture of (K) was subjected to anti-peptide II and ACTIN specific immunoblot. (M) A sister culture of (K) was immunohistochemically stained with anti-peptide II and hematoxylin. All bars represent 20 µm. (*) changes of LINC013026-68AA migration in SDS-PAGE induced by phosphorylation.