Recombinant Human IMMT protein, His-tagged

• Cat.No.: IMMT-838H
• Product Overview: Recombinant Human IMMT fused with His tag at N-terminal was expressed in E. coli.

Specification

• Species: Human
• Source: E.coli
• Tag: His
• Description: Mitochondrial inner membrane protein is a protein that in humans is encoded by the IMMT gene.
• Form: 25mM Tris, pH8.0, 150 mM NaCl, 10% glycerol, 100 mM Arg.
• Molecular Mass: 83.7 kDa
• Purity: > 80% as determined by SDS-PAGE and Coomassie blue staining
• Storage: Store at -80 centigrade. Avoid repeated freeze-thaw cycles. Stable for at least 3 months from receipt of products under proper storage and handling conditions.
• Concentration: >50 ug/mL as determined by microplate BCA method
• Publications:
  Global Interactome Mapping of Mitochondrial Intermembrane Space Proteases Identifies a Novel Function for HTRA2 (2019)
  Interactome Mapping of the Mitochondrial Intermembrane Space Proteases Identifies a Novel Function of HTRA2 (2020)

Gene Information

• Gene Name: IMMT inner membrane protein, mitochondrial [ Homo sapiens ]
• Official Symbol: IMMT
• Synonyms: IMMT; inner membrane protein, mitochondrial; inner membrane protein, mitochondrial (mitofilin); mitochondrial inner membrane protein; HMP; MINOS2; mitochondrial inner membrane organizing system 2; mitofilin; P87; P89; motor protein; proliferation-inducing gene 4; cell proliferation-inducing protein 52; cell proliferation-inducing gene 4/52 protein; PIG4; PIG52; P87/89; MGC111146; DKFZp779P1653
• Gene ID: 10989
• mRNA Refseq: NM_006839
• Protein Refseq: NP_006830
• MIM: 600378
• UniProt ID: Q16891
• Chromosome Location: 2p11.2
• Function: protein binding

Citation

A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

Journal: Acta Neuropathologica    PubMed ID: 36104602    Data: 2022/9/14

Authors: Yutong Shang, Xiaoyan Sun, Xin Qi

Article Snippet:The Alexa 488/568/405/633 fluorescent secondary antibodies were from Invitrogen.The Alexa 488/568/405/633 fluorescent secondary antibodies were from Invitrogen.. CHCHD3 (CHCHD3-195H) and Mitofilin (IMMT-838H) recombinant proteins were from Creative BioMart (Shirley, NY, USA).. CHCHD6 recombinant protein (TP302975) was from Origene (Rockville, MD, USA).CHCHD6 recombinant protein (TP302975) was from Origene (Rockville, MD, USA).

CHCHD6 selectively decreases at transcription level in AD models. a Mitochondrial fractions were isolated from APP stable Neuro2a cells and subjected to Blue Native PAGE (BN-PAGE) analysis followed by western blotting (WB). MICOS complex level was detected with antibody against CHCHD6. ATPB was analyzed by SDS-PAGE followed by WB, using as a loading control. Left: representative blots from 5 independent experiments. Right: relative density of CHCHD6-immunoreactive band around 720 kDa in contrast to ATPB. b Total lysates harvested from APP stable Neuro2a cells were subjected to WB with the indicated antibodies. Left: representative blots from 3 independent experiments. Right: relative density of CHCHD6, Mitofilin and CHCHD3 in contrast to actin. c RNA was extracted from APP stable Neuro2a cells. The expression of MICOS complex components was analyzed by qPCR. Heat map analysis shows the mean of the genes analyzed. n = 6 independent experiments for CHCHD10, and n = 4 independent experiments for others. **** p < 0.0001 (Con vs. APP wt or APP swe cells). d Mitochondrial fractions were isolated from the hippocampus of WT, APP NL?G?F and APP NL?F mice at the ages of 3, 6, and 9 months, and subjected to BN-PAGE analysis. Histogram: relative density of CHCHD6-immunoreactive band around 720 kDa in contrast to ATPB. n = 4 mice/group. e The hippocampus of WT, APP NL?G?F and APP NL?F mice was harvested at the ages of 3, 6, and 9 months. Total protein levels of CHCHD6, Mitofilin and CHCHD3 were examined by WB. n = 4 mice/group. f RNA was extracted from the hippocampus of 6-month-old APP NL?G?F mice or 9-month-old APP NL?F mice and age-matched wild-type (WT) mice. The expression of MICOS complex components was analyzed by qPCR. Heat map analysis shows the mean of the genes analyzed. n = 9 mice/group for CHCHD3 of APP NL?G?F mice, and n = 5 mice/group for other groups. * p < 0.05 (WT vs. APP NL?G?F mice or APP NL?F mice). g RNA was extracted from the postmortem hippocampus of AD patients and control subjects. The expression of CHCHD6, Mitofilin and CHCHD3 was analyzed by qPCR. n = 9 individuals/group. All data are presented as mean ± SEM and were compared using one-way ANOVA with Tukey’s post hoc test in ( a – e ), and unpaired Student’s t test in ( f – g )

CHCHD6 and APP are interdependent to regulate each other. a . The total protein lysates of control and CHCHD6 KO HT-22 cells were subjected to immunoprecipitation (IP) with anti-APP antibody, followed by WB. Shown blots are representative of three independent experiments. b APP recombinant protein (500 ng) was incubated with either CHCHD6, Mitofilin or CHCHD3 recombinant proteins (500 ng, each). Immunoprecipitates with anti-APP antibodies was analyzed by immunoblotting with the indicated antibodies. Data are representative of 2 independent experiments. Control and CHCHD6 KO HT-22 cells were stained with anti-APP and anti-CHCHD6 antibodies ( c ) or anti-APP and anti-FACL4 antibodies ( g ), and subjected to in situ Duolink proximity ligation assay (PLA) analysis. Histogram shows the number of PLA-positive puncta (red). At least 47 cells/group (for APP and CHCHD6) and 250 cells/group (for APP and FACL4) were analyzed. The data were from 3 independent repeats. Brain sections from 6-month-old WT and APP NL?G?F mice ( n = 4 mice/group) were stained with anti-APP and anti-CHCHD6 antibodies ( d ) or anti-APP and anti-FACL4 antibodies ( h ), and subjected to PLA analysis. Histogram: the number of PLA-positive puncta (red) was quantified from at least four separate fields of each sample. e The total protein lysates of hippocampus of control subjects and AD patients were subjected to immunoprecipitation (IP) with anti-APP antibody, followed by WB. Shown blots are representative of three independent experiments. f HEK293 cells were infected with control or APP shRNA lentivirus. Total protein lysates were analyzed by WB with indicated antibodies. Histograms: relative density of CHCHD6 to actin. n = 6 independent experiments. i Control and CHCHD6 KO HT-22 cells were transfected with Flag empty vector (Flag-EV), or Flag-tagged CHCHD6 (D6-Flag) plasmids for 2 days. The total protein lysates were subjected to WB with the indicated antibodies. Histogram: the relative density of C99 and APP to actin. n = 3 independent experiments for C99 and n = 4 independent experiments for APP. j Control and CHCHD6 KO HT-22 cells were stained with anti-AICD antibody and Hoechst probe. The intensity of AICD was quantified and shown in the histogram. At least 300 cells/group were analyzed, and the data were from 4 independent experiments. k HEK293 cells were infected with control or APP shRNA lentivirus, and then transfected with the indicated plasmids for 72 h. The total protein lysates were subjected to WB. Red arrow: AICD. Histogram: the relative density of CHCHD6 to actin. n = 4 independent experiments. l HEK293 cells were transfected with CHCHD6 promoter-luc along or together with AICD, and/or Fe65 and/or Tip60 for 36 h. Cells were lysed, and the luciferase activity was measured. n = 8 for control and n = 4 for other groups. m HEK293 cells were transfected with GFP-labeled AICD, myc-Fe65 and Tip60-flag. CHIP analysis on CHCHD6 promoter was carried out with indicated antibodies. Hes1 gene ChIP is reported as a control. n = 2 independent experiments. All the data are mean ± SEM. The data in panel c , d , f – h , j were compared by the unpaired Student’s t test , and the data in panels i , k and l were compared by one-way ANOVA with Tukey’s post hoc test