length cd44  (ATCC)

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades CD44 through the lysosome pathway. ( A ) Immunoblots to detect decreased CD44 and increased BAX, BID, and MARCH8 expression levels in MARCH8-overexpressing cells compared to GFP control cells. ( B ) Immunofluorescence staining with anti-CD44 antibody showing decreased expression of membrane protein CD44 (blue) in MARCH8-GFP expressing cells. ( C ) Flow cytometry histogram overlay (left panel) and dot plots (right panels) indicating MARCH8-decreased CD44 expression levels in negative association with MARCH8-GFP signals. ( D ) Immunoblots of endogenous CD44 and exogenous MARCH8-GFP after transient transfection of MARCH-GFP and treatment with MG-132 or chloroquine (CLQ) to block the proteasomal or lysosomal degradation pathways, respectively. ( E ) Immunoblots of MARCH8 and FLAG-tagged CD44 after anti-FLAG mediated immunoprecipitation (IP) of the lysates of HEK-293 cells after transfections with MARCH8-GFP and CD44-FLAG (standard isoform CD44s and full-length CD44f) and treatment with CLQ, indicating the interactions between MARCH8 and CD44 (CD44s or CD44f). ( F ) Immunoblots of BAX, BID, CD44, and MARCH8 in the MDA-MB-231 cells with stable expression of GFP or MARCH8-GFP with transient transfection of a FLAG vector control or restoration of CD44 expression via CD44s-FLAG. CD44 overexpression slightly inhibited the expression of proapoptotic BID and BAX in MARCH8-GFP-overexpressing cells.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Transfection, Blocking Assay, Immunoprecipitation, Plasmid Preparation, Over Expression

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades STAT3 through the proteasome pathway. ( A ) Immunoblots of AKT, pAKT, ERK, and pERK in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, both adherent and in suspension. ( B ) Immunoblots of STAT3, pSTAT3 (Y705), CD44, and MARCH8 in GFP- and MARCH8-GFP-expressing cells in the absence or presence of MG-132 and CLQ, both adherent and in suspension. ( C ) Immunoblots of MARCH8, STAT3, pSTAT3 (Y705), and ubiquitin in lysates of cells co-transfected with STAT3 (wildtype or Y705F) and MARCH8-GFP (or GFP control) and immunoprecipitated by anti-STAT3 and anti-pSTAT3 (Y705). ( D ) Immunoblots of CD44, STAT3, BID, and BAX in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, with transient transfection with CD44, STAT3, or mutant Y705F as indicated, both adherent and in suspension.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Mutagenesis