antibody cct6  (Santa Cruz Biotechnology)

Journal: The Journal of Clinical Investigation

Article Title: ANKRD55 is a key regulator of T cell inflammation in multiple sclerosis

doi: 10.1172/JCI195214

Figure Lengend Snippet: ( A ) Overexpression of ANKRD55 and TCP1 in HEK293T cells followed by co-IP analysis to determine their interaction. ( B ) Protein extraction from Jurkat cells with subsequent co-IP assay to determine the interaction between ANKRD55 and TCP1. ( C ) PLA experiment in Jurkat cells to assess the interaction between ANKRD55 and TCP1. Cells were fixed and incubated with primary antibodies against ANKRD55 and TCP1, followed by PLA probe ligation and amplification. Red fluorescent puncta indicate close proximity (<40 nm) between ANKRD55 and TCP1, suggesting a direct or complex-mediated interaction. Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bars: 2 μm (top), 5 μm (bottom). ( D ) Immunofluorescent staining of Jurkat cells to analyze the colocalization of ANKRD55 and TCP1. Scale bars: 2 μm. ( E – K ) Co-IP assays were performed using HEK293T cells to investigate the interactions between ANKRD55 and individual CCT subunits, including CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, and CCT8. ( L ) Immunofluorescent staining of Jurkat cells to assess colocalization between ANKRD55 and specific CCT subunits (CCT2, CCT3, CCT4, and CCT7). Scale bars: 2 μm (first 4 panels), 1 μm (fifth panel). ( M ) Immunofluorescent staining of Jurkat cells to visualize the subcellular localization of ANKRD55, TCP1, and pericentrin. Scale bars: 5 μm. ( N – P ) Immunoblotting analysis of TCP1 ( N ), CCT3 ( O ), and CCT6 ( P ) expression levels in Jurkat cells following ANKRD55 knockdown.

Article Snippet: TCP1 antibody (catalog 10320 and 68183), CCT2 antibody (catalog 68214), CCT3 antibody (catalog 60264), CCT4 antibody (catalog 67455), CCT5 antibody (catalog 67400), CCT6 antibody (catalog 19793), CCT7 antibody (catalog 68214), DYKDDDDK tag polyclonal antibody (catalog 20543), HA tag polyclonal antibody (catalog 51064), phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibody (catalog 28733), CD247 polyclonal antibody (catalog 12837), and phospho-LCK-Y394 rabbit antibody (catalog AP0182) were purchased from Proteintech.

Techniques: Over Expression, Co-Immunoprecipitation Assay, Protein Extraction, Incubation, Ligation, Amplification, Staining, Western Blot, Expressing, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: ANKRD55 is a key regulator of T cell inflammation in multiple sclerosis

doi: 10.1172/JCI195214

Figure Lengend Snippet: ( A and B ) α-Tubulin immunoblotting in lysates and pellet determined by microtubule sedimentation assay in Jurkat cells with TCP1 ( A ) or ANKRD55 ( B ) knocked down. ( C ) TCP1 degradation rate analyzed via immunoblot after cycloheximide (CHX; 70 μM) treatment for 0–48 hours in control and Jurkat cells overexpressing ANKRD55. ( D – F ) Co-IP detection of interactions between CCT5 and TCP1 ( D ), CCT3 ( E ), or CCT6 ( F ) at varying concentrations of ANKRD55 in HEK293T. ( G ) Immunofluorescence analysis of immune synapse formation between Jurkat and Raji cells. Jurkat cells were prelabeled with CMAC. Raji cells were stimulated with SEE for 30 minutes. The 2 cell types were then cocultured for 30 minutes. Cells were stained with antibodies against ANKRD55, TCP1, pericentrin, and α-tubulin to visualize protein localization at the immune synapse. Scale bars: 2 μm. BF, bright-field; CMAC, CellTracker blue fluorescent probe. ( H ) Flow cytometry–based immune synapse (IS) pattern analysis. ( I and J ) Raji cells (APCs) stained with CFSE and stimulated with SEE for 30 minutes at 37°C and Jurkat cells (T cells) stained with CMTPX. T cell conjugation with APCs after 20 minutes of contact was analyzed by flow cytometry. Conjugate percentages were determined for Jurkat cells with ANKRD55 or TCP1 knocked down ( I ) and pretreatment with HSF1A (50 μM) for 2 hours ( J ). ( K ) Mean clinical score of EAE in mice injected intraperitoneally with PBS or HSF1A (20 mg/mL) ( n = 7 or 8 mice per group), induced by active immunization with MOG 35–55 . ( L ) Immunoblot analysis of TCR signaling in Jurkat cells. Cells included vector control, a stable ANKRD55-overexpressing cell line, and ANKRD55-overexpressing cells pretreated with HSF1A (50 μM, 2 hours). All groups were stimulated on plates coated with anti-CD3 and anti-CD28. Lysates were collected at the indicated time points (1, 2, 15, and 30 minutes) and probed for TCR signaling–associated proteins. ( M ) H&E and Luxol fast blue (LFB) staining of spinal cord sections at the peak of EAE disease. Arrows indicate areas of demyelination. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, by 2-way ANOVA with Tukey’s multiple-comparison test. Data are shown as mean ± SEM.

Article Snippet: TCP1 antibody (catalog 10320 and 68183), CCT2 antibody (catalog 68214), CCT3 antibody (catalog 60264), CCT4 antibody (catalog 67455), CCT5 antibody (catalog 67400), CCT6 antibody (catalog 19793), CCT7 antibody (catalog 68214), DYKDDDDK tag polyclonal antibody (catalog 20543), HA tag polyclonal antibody (catalog 51064), phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibody (catalog 28733), CD247 polyclonal antibody (catalog 12837), and phospho-LCK-Y394 rabbit antibody (catalog AP0182) were purchased from Proteintech.

Techniques: Western Blot, Microtubule Sedimentation Assay, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Conjugation Assay, Injection, Plasmid Preparation, Comparison

Journal: Cell Stress & Chaperones

Article Title: Co-expression of CCT subunits hints at TRiC assembly

doi: 10.1007/s12192-019-01028-5

Figure Lengend Snippet: A model of CCT subunit arrangement based on hetero-oligomerization data and the known CCT subunit order. Starting from CCT5 homo-oligomers, this theoretical model would show sequential subunit exchange to finally form the known CCT subunit order (Leitner et al. 2012). In the first step, CCT2 enters one ring (our data: CCT5 interacts with CCT2). Since CCT2 forms intra-ring contacts, another CCT2 will enter the opposite ring (our data: CCT5 interacts with CCT2; final structure: CCT2 forms homotypic contacts with itself while being adjacent to CCT5). Next, CCT4 forms inter-ring contacts with CCT2 and CCT5 (our data: CCT5 interacts with CCT2 and CCT4; final structure: CCT4 is adjacent to CCT2). Now, CCT1 can form inter-ring contacts with CCT4 (our data: CCT5 interacts with CCT1 and CCT2; final structure: CCT1 is adjacent to CCT4). Subsequently, CCT3 forms inter-ring contacts with CCT4 while being adjacent to CCT1 and CCT1 forms intra-ring contacts with CCT7 (our data: CCT5 interacts with CCT2, CCT3, and CCT7; final structure: CCT3 is adjacent to CCT1, while CCT7 is on top of CCT1 while being adjacent to CCT5). Next, CCT8 forms intra-ring contacts with CCT3 while being adjacent to CCT7 (our data: CCT5 interacts with CCT2, CCT3, CCT7, and CCT8; final structure: CCT8 is adjacent to CCT7 while being on top of CCT3). Finally, CCT6 takes its place between CCT3 and CCT8 while making homotypic contacts with itself (our data: CCT6 does not interact with CCT5 nor CCT4; final structure: CCT6 forms homotypic contacts with itself while being adjacent to CCT3 and CCT8)

Article Snippet: The primary antibodies were from Santa Cruz Biotechnology: CCT1, sc-53454; CCT2, sc-28556; CCT3, sc-33145; CCT4, sc-137092; CCT5, sc-13886; CCT6, sc-100958; CCT7, sc-130441; and CCT8, sc-13891.

Techniques: