Human TREM2 Antibody

Detection of Human TREM2 by Western Blot

TREM2 is shed at a single cleavage site in the ectodomain between histidine 157 and serine 158Regulated intramembrane proteolysis of TREM2. ADAM10 initiates proteolytic processing of TREM2 by liberating its ectodomain (sTREM2). Subsequent processing of the membrane retained C‐terminal fragment (CTF) by gamma‐secretase within the transmembrane domain (TM) releases the intracellular domain (ICD) into the cytosol. A short p3‐like peptide (Haass et al, 1993) may be secreted.Outline of strategy I for mass spectrometric (MS) determination of the N‐terminal end of the TREM2 CTF enriched upon gamma‐secretase inhibition using DAPT.Western blot analysis of TREM2 stably expressed in HEK293 Flp‐In cells upon gamma‐secretase inhibition using DAPT. Application of DAPT leads to a robust accumulation of the TREM2 CTF under constitutive conditions as well as upon phorbol 12‐myristate 13‐acetate (PMA) mediated stimulation of TREM2 ectodomain shedding. The TREM2 9D11 antibody raised against the TREM2 C‐terminus was used to detect TREM2, and calnexin levels were analyzed as a loading control. Here, as in all other Western blots in Figs 1 and 2, the two lanes represent samples from two separate wells seeded at the same time.MALDI‐TOF MS determination of the ectodomain cleavage site by immunoprecipitation of TREM2 CTF. The peak at 8,841.48 Da corresponds to a single cleavage site between histidine 157 and serine 158. Very minor additional peaks may represent cellular degradation products, as the N‐terminal counterpart cannot be observed (see Fig 1I).Stimulation of ectodomain shedding by PMA leads to strong increases in sTREM2 (anti‐HA, upper panel) and sAPP alpha (2D8, lower panel). DMSO served as a vehicle control. EV, empty vector control.MALDI‐TOF MS determination of the ectodomain cleavage site upon ADAM17 stimulation using PMA. TREM2 CTFs were enriched by gamma‐secretase inhibition using DAPT (Fig 1C). The peak at 8,837.09 Da corresponds to a single cleavage site between histidine 157 and serine 158.Outline of strategy II for MS determination of the C‐terminal end of sTREM2 generated by ectodomain shedding.Western blot analysis of full‐length TREM2‐TEVFlag (middle panel) and ectodomain shedding (upper panel) upon transient transfection of HEK293 Flp‐In cells. EV, empty vector control.MALDI‐TOF MS determination of the ectodomain cleavage site following the strategy outlined in Fig 1G. The peak at a mass of 3,947.78 Da corresponds to cleavage C‐terminal of histidine 157 and is absent in cells transfected with an empty expression vector.TREM2 is shed predominantly C‐terminal to histidine 157 as observed in two different cell types as well as under constitutive and PMA‐stimulated conditions.Western blot analysis of full‐length TREM2‐TEVFlag (middle panel) and ectodomain shedding (upper panel) upon transient transfection of THP‐1 monocytes.MALDI‐TOF MS determination of the ectodomain cleavage site in THP‐1 monocytes following the strategy outlined in Fig 1G. The peak at a mass of 3,943.80 Da corresponds to cleavage C‐terminal of histidine 157 and is absent in cells transfected with an empty expression vector.Source data are available online for this figure. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28855300), licensed under a CC-BY license. Not internally tested by R&D Systems.