Figure 3
Alternative splicing of ALCAM introduces sensitivity to MMP14-dependent shedding. (a) ALCAM ELISA for quantification of basal extracellular domain shedding in parental HT1080 cells (Par) overexpressing ALCAM-Iso1 (Iso1-OE) or ALCAM-Iso2 (Iso2-OE), as well as in ALCAM-KO HT1080 cells (KO) rescued with either ALCAM-Iso1 (KO + Iso1) or ALCAM-Iso2 (KO + Iso2). ALCAM expression was measured in whole cell lysate (WCL) and shedding was measured in conditioned medium (CM). P-values were calculated using Kruskal-Wallis test with Dunn’s post-test; ns: not significant, *P < 0.05, ****P < 0.0001. (b) Immunoblot analysis of ALCAM expression and basal extracellular domain shedding in indicated cells. ICD: intracellular domain, ECD: extracellular domain. ALCAM fragments generated by shedding are marked as follows: 20 kDa intracellular domain fragment (ICD-20, 
), 58 kDa extracellular domain fragment (ECD-58, 
), 55 kDa extracellular domain fragment (ECD-55, 
), and 40 kDa extracellular domain fragment (ECD-40, 
). (c) Schematic of antibody epitopes and cleavage sites in ALCAM-Iso1 and ALCAM-Iso2. (d) Immunoblot analysis of extracellular domain shedding of ALCAM-KO HT1080 cells expressing ALCAM-Iso1 (KO + Iso1) or ALCAM-Iso2 (KO + Iso2) treated with an ADAM17-specific inhibitor, Compound 32 (Cmpd32, 10 μM), or a global metalloprotease inhibitor, GM6001 (10 μM). (d’) Changes in shedding were quantified as a ratio between intact ALCAM (WCL), and shed ALCAM (CM), with the untreated control normalized to 1. (e) Immunoblot analysis of extracellular domain shedding of ALCAM-KO HT1080 cells expressing ALCAM-Iso2 (KO + Iso2) transfected with anti-MMP14 siRNA. Results are representative of two independent experiments. (e’) Changes in shedding were quantified as percent decrease in ECD-40 band density compared to control (siGRP), or ratio of ECD-40 density to ECD-55 density. (e”) Knockdown (KD) of MMP14 was quantified as percent decrease in band density compared to control (siGRP). (b,d,e) 5 min exposures are shown. Individual blots are outlined in black. WCL blots were redeveloped with HRP-tagged anti-GAPDH for loading control. Blots have been cropped to show relevant bands, full-length blots are available in Supplementary Figure S7. (d’,e’,c’) Samples were derived from the same experiment. Gels and blots were processed in parallel.
