The -tubulin SUMOylation in a specific type of cell or tissue may be controlled by the level and activity of all the SUMOylation machinery proteins for -tubulin, such as E1, E2, E3 (if any), and SUMO-specific peptidase 1 (SENP1), and is compatible with distinct cell property or tissue function. two up-shifted poor bands were observed in cells expressing Flag-SUMO2 or Flag-SUMO3 (Physique?1A). Consistently, SUMOylated bands were observed in -tubulin immunoprecipitates when probed by SUMO1 Ab (Physique?1B). In addition, in HEK293 cells transfected with HA-Ubc9, we found that -tubulin could be coimmunoprecipitated with Ubc9, the unique E2 enzyme for SUMOylation (Physique?1C), indicating that -tubulin interacts with the SUMOylation machinery. All above evidence suggested that -tubulin is usually a SUMO1-altered substrate in cells. To further validate the SUMOylation of -tubulin by SUMO1, SUMOylation assay using brain tubulin as substrates was performed. Immunoblotting showed that -tubulin was SUMOylated in the presence of recombinant SAE1/2, Ubc9, and SUMO1GG (Physique?1DCH), with the ratio of SUMOylated -tubulin to unSUMOylated being 7.8% (Figure?1G). Further SUMOylation using MTs and tubulin dimers as substrates showed that -tubulin in dimers could be more efficiently SUMOylated than that in MTs (Physique?1I), suggesting that -tubulin SUMOylation is a soluble-tubulin-enriched PTM. We also surveyed the SUMOylation of -tubulin in several cell I-BRD9 lines and mouse tissues, and found that the level and pattern of -tubulin SUMOylation varied a lot across cell lines and mouse tissues investigated (Supplementary Physique S1A and B). The -tubulin SUMOylation in a specific type of cell or tissue may be controlled by the level and activity of all the SUMOylation machinery proteins for -tubulin, such as E1, E2, E3 (if any), and SUMO-specific peptidase 1 (SENP1), and is compatible with unique cell house or tissue function. These data show that -tubulin is able to be SUMOylated and SUMOylation assay using purified GST-SAE2/1, GST-Ubc9, His-SUMO1GG, and brain tubulins. (G) Ratio of density of SUMOylated bands to unSUMOylated bands. (H) Purified tubulin was SUMOylated and probed with -tubulin Ab. (I) SUMOylation assay using soluble tubulins and MTs. (J) Immunoprecipitates with -tubulin Ab from HEK293 cells expressing Flag-SUMO1, Flag-SUMO2, or Flag-SUMO3 were probed with Flag and -tubulin Abdominal muscles. (K) Purified tubulin was SUMOylated and probed with -tubulin Ab. (L) Endogenous -tubulin in HEK293 cells was immunoprecipitated and probed with SUMO1 Ab. The experiments were repeated three times. Because /-tubulin constitutively exist as dimers, whether -tubulin could be SUMOylated was examined. We found that upon SUMO1, SUMO2, or SUMO3 overexpression, -tubulin was mainly altered by SUMO1 in HEK293 cells (Physique?1J). In addition, -tubulin could be SUMOylated (Physique?1K). However, SUMOylation of endogenous -tubulin in cells without SUMO1 overexpression was almost undetectable (Physique?1L). Since the basal level I-BRD9 of -tubulin SUMOylation is usually low in cells, we mainly focused on the study of -tubulin SUMOylation. SUMOylation is mainly enriched in soluble -tubulin To determine the localization of SUMOylated Mouse monoclonal to CD29.4As216 reacts with 130 kDa integrin b1, which has a broad tissue distribution. It is expressed on lympnocytes, monocytes and weakly on granulovytes, but not on erythrocytes. On T cells, CD29 is more highly expressed on memory cells than naive cells. Integrin chain b asociated with integrin a subunits 1-6 ( CD49a-f) to form CD49/CD29 heterodimers that are involved in cell-cell and cell-matrix adhesion.It has been reported that CD29 is a critical molecule for embryogenesis and development. It also essential to the differentiation of hematopoietic stem cells and associated with tumor progression and metastasis.This clone is cross reactive with non-human primate -tubulin, proximity ligation assay (PLA), which enables detection of protein modification (Soderberg et al., 2006), was performed using main antibodies against -tubulin and SUMO1. Immunostaining showed that PLA signals were located both on MTs and in the cytoplasm, but largely (70%) distributed in the cytoplasm (Physique?2A and B), suggesting that SUMOylation of -tubulin may mainly occur on unpolymerized tubulins. To further confirm this phenomenon, soluble and polymerized tubulins were separated in HEK293 cells expressing Flag-SUMO1. Followed SUMOylation detection of these two pools by IP showed I-BRD9 that, in line with the PLA results, soluble -tubulin experienced a much higher level of SUMOylation than polymerized -tubulin (Physique?2C and D). The preferential distribution on soluble -tubulin in cells was consistent with a higher catalytic efficiency of SUMOylation machinery toward soluble tubulins (Physique?1I). Open in a separate windows Physique 2 SUMOylation is mainly enriched in soluble -tubulin. (A) PLA with -tubulin and SUMO1 Abdominal muscles was performed in HEK293 cells. Confocal images of PLA signals and tubulin labelled after PLA are shown. The enlarged image of the boxed area is usually shown at the lower right. Scale bar, 10?m. (B) PLA dots on and off MTs were quantified. and mice.