Journal: JCI Insight

Article Title: LMAN1 serves as a cargo receptor for thrombopoietin

doi: 10.1172/jci.insight.175704

Figure Lengend Snippet: ( A ) Plasma TPO level is reduced in LMAN1-deficient compared with WT littermate control mice. ( B ) Tpo mRNA levels were indistinguishable between Lman1 –/– and WT control mice, as demonstrated using 3 different Tpo primer sets. ( C ) FLAG-tagged LMAN1 (LMAN1-FLAG) and myc-tagged TPO (TPO-myc) were expressed in HEK293T cells. A physical interaction between TPO and LMAN1 was suggested, as an anti-FLAG antibody coimmunoprecipitated TPO-myc. In, input (10%); IP, immunoprecipitated fraction. ( D and E ) A reporter human HEK293T cell line that expresses EGFP-fused TPO and mCherry-fused A1AT was generated. ( D ) Deletion of LMAN1 using an LMAN1 -targeting sgRNA resulted in intracellular accumulation of TPO and A1AT compared with cells transduced with a nontargeting (NT) sgRNA. ( E ) Immunofluorescence microscopy demonstrates significantly increased colocalization of TPO in the ER (labeled with blue fluorescent protein) in cells transfected with LMAN1 -targeting sgRNA (LMAN1 KO) compared with control cells (WT). **** P < 0.0001. PCC, Pearson correlation coefficient. ( F ) A reporter cell line expressing TPO fused to mCherry and PCSK9 fused to EGFP was generated. LMAN1 deletion results in intracellular accumulation of TPO but not PCSK9. ( G ) LMAN1 deletion (LMAN1 KO) in HEP3B cells treated with 80 ng/mL IL-6 results in reduced TPO in the supernatant compared with control cells transduced with nontargeting (NT) sgRNA. *** P < 0.001. Statistical analyses in this figure were performed using unpaired Student’s t test.

Article Snippet: LMAN1 immunoprecipitation was performed using anti-FLAG antibody covalently bonded to agarose beads (EZview Red Anti-FLAG M2 affinity gel, MilliporeSigma).

Techniques: Clinical Proteomics, Control, Immunoprecipitation, Generated, Transduction, Immunofluorescence, Microscopy, Labeling, Transfection, Expressing