TABLE 1.
Major studies on identifying Sigmar1’s subcellular organelle localization (in chronological order).
| Subcellular organelles | Experimental methods | Cells or Tissues | Antibody and antigen | Antibody validation | References |
| Mitochondria Microsomal Synaptosomal fractions | Western blotting on subcellular fractions | Rat brain | Anti-Sigmar1 Rabbit Polyclonal antibody raised against synthetic amino acid residues 143–163 of Sigmar1 (produced by authors) | Not listed | Yamamoto et al., 1999 |
| Plasma membrane Mitochondrial membranes Vesicles or elongated cisternae of ER | Immunoelectron microscopy | Adult rat brain hypothalamus and hippocampus sections | Anti-Sigmar1 Rabbit Polyclonal IgG raised against synthetic peptide of Sigmar1 amino acid sequence 143–162 (produced by authors) | Antibody specificity confirmed through absence of staining in brain tissue sections when co-incubated with Sigmar1 specific antigen | Alonso et al., 2000 |
| Perinuclear areas Plasmalemmal regions of cell-cell contact Growth cones of neurites | Fluorescence microscopy of co-immunocytohistochemistry and co-immunoprecipitation of endogenous Sigmar1 with Ankyrin B and IP3R3 receptors | Mouse neuroblastoma x rat glioma hybrid NG-108-15 cells | Anti-Sigmar1 Rabbit antibody raised against N-terminal residues of mouse Sigmar1 (produced by authors) | Not listed | Hayashi and Su, 2001 |
| Mitochondrial membranes | i. Radioisotope labeled Sigmar1 agonist [3H](+)-pentazocine binding assay ii. Immunofluorescence | i. Isolated rat liver mitochondria ii. Rat liver sections | Anti-Sigmar1 Rabbit Polyclonal IgG (Alonso et al., 2000) | Validated by original contributors at (Alonso et al., 2000) | Klouz et al., 2003 |
| Plasma membranes | i. Exogenous expression of N-terminal and C-terminal GFP tagged Sigmar1 constructs and fluorescence microscopy ii. Co-immunoprecipitation with anti-Kv1.4 antibody | i. Xenopus Oocytes ii. Triton X-100 solubilized membrane lysates of rat pituitary gland | Anti-Sigmar1 Rabbit Polyclonal IgG (Yamamoto et al., 1999) | Not listed | Aydar et al., 2002 |
| i. ER associated detergent insoluble lipid droplets/microdomains ii. Sigmar1 immunostained reticular structure was noted negative for endosomes (EEA-1), mitochondria (Mitotracker, bcl-2), lysosomes (Lyso Tracker, LAMP-1), synaptic vesicles (Synapsin II), plasma membrane (Fas and CTx-B), golgi (GM130), ER-associated proteins | Fluorescence microscopy; cells were specially treated with 0.02% SDS for 10 min | NG-108 cells (Hayashi and Su, 2003a, b), Rat hippocampal differentiated oligodendrocytes culture (Hayashi and Su, 2004) | i. Exogenously expressed C-terminal Enhanced Yellow Fluorescent Protein (EYFP)-tagged mouse Sigmar1 construct ii. Polyclonal rabbit anti-guinea pig Sigmar1 antibody raised against guinea pig Sigmar1 amino acid sequence 144–165 (Hayashi and Su, 2003b). | i. Exogenous overexpression ii. Anti-Sigmar1 staining pattern have been visually compared to expression pattern of exogenously expressed Sigmar1-EYFP plasmid construct (Hayashi and Su, 2003a, b) | Hayashi and Su, 2003a, b, 2004 |
| Mitochondria Mitochondria-associated ER membranes (MAM) Nuclear and microsomal fractions | i. Immunofluorescence staining of endogenous Sigmar1 in cells transfected with Mitochondria and ER fluorescent constructs, ii. Western blot analysis on ultracentrifugation based subcellular fractionations | Chinese hamster ovary (CHO) cells | Anti-Sigmar1 Rabbit antibodies raised against amino acid residues 52–69 and 143–165 of Rat Sigmar1 (produced by authors) | Western blot on CHO cell lysates and on different rat organs | Hayashi and Su, 2007 |
| Focal adhesion contacts (FACs) | Co-localization of Sigmar1 with FAC protein Talin in immunofluorescence staining | CHO-K1 cells | Anti-Sigmar1 Rabbit antibody raised against Maltose Binding Protein (MBP)-Guinea Pig full length Sigmar1 fusion protein (Ramachandran et al., 2007) (produced by authors) | Western blot analysis using guinea pig Sigmar1 overexpressing COS-7 cells as positive control (Ramachandran et al., 2007) | Mavlyutov and Ruoho, 2007 |
| i. Motor neurons of mouse brain Medulla and spinal cord ii. Cholinergic post-synaptic terminals co-localized to Kv2.1 potassium channels iii. Putative subsurface ER cisternae close to plasma membrane | i. Immunohistochemistry ii. Co-immunofluorescence with cholinergic neuron markers iii. Immuno-electron microscopy | Mouse brain and spinal cord sections | Anti-Sigmar1 Rabbit antibody raised against Maltose Binding Protein (MBP)-Guinea Pig full length Sigmar1 fusion protein (Ramachandran et al., 2007) | Sigmar1 knockout mice brain sections as negative control in immunohistochemistry | Mavlyutov et al., 2010, 2012 |
| Mitochondria ER | Fluorescence microscopy | Mouse neuroblastoma Neuro-2a cells | Exogenously expressed eGFP and mCherry tagged Sigmar1’s correlative, visual colocalization observation with erRFP and mtGFP labeled ER and mitochondria, respectively | Exogenous overexpression | Shioda et al., 2012 |
| Mitochondria and MAM fractions | Western blot analysis on subcellular fractions | Chinese hamster ovary (CHO) cells | Anti-Sigmar1 Rabbit antibodies raised against Rat Sigmar1 (Hayashi and Su, 2007) | Western blot on CHO cell lysates and on different rat organs (Hayashi and Su, 2007) | Mori et al., 2013 |
| ER and Nuclear Envelope | Confocal immunofluorescence staining | NG108 and Neuro-2a cells | Anti-Sigmar1 (Santa Cruz Biotechnology) | Commercial vendor | Tsai et al., 2015 |
| Predominantly in nuclear envelope, sparsely at ER cisternae located subsurface of plasma membranes | Immuno-electron microscopy | Mouse retinal photoreceptor cells | Anti-Sigmar1 Rabbit antibody raised against Maltose Binding Protein (MBP)-Guinea Pig full length Sigmar1 fusion protein (Ramachandran et al., 2007) | Reported at (Ramachandran et al., 2007) | Mavlyutov et al., 2015b |
| Plasma membrane ER Nuclear envelope | Immuno-electron microscopy | Mouse and rat dorsal root ganglion (DRG) cells | Anti-Sigmar1 Rabbit antibody raised against Maltose Binding Protein (MBP)-Guinea Pig full length Sigmar1 fusion protein (Ramachandran et al., 2007) | Validated through absence of immunofluorescence staining using Sigmar1 knockout mouse DRG tissue | Mavlyutov et al., 2016 |
| Nucleus Nucleoplasmic reticulum Plasma membrane subsurface ER cisternae | Immuno-electron microscopy | NSC34 cells (produced by fusion of motor neuron enriched, embryonic mouse spinal cord cells with mouse neuroblastoma), Sigmar1-null NSC34 cells | Endogenous Sigmar1 stained with antibody as reported in Ramachandran et al. (2007), Exogenously expressed full length Sigmar1-GFP-APEX2 (ascorbate peroxidase 2) fusion construct were enhanced using 3, 3′-diaminobenzidine (DAB) incubation | Reported at (Ramachandran et al., 2007) | Mavlyutov et al., 2017 |
| i. ER, Mitochondria associated ER membranes (MAM) ii. Cholesterol-containing giant unilamellar vesicles (GUVs) | Immuno-fluorescence microscopy | HEK293T cells | i. Exogenously expressed GFP-tagged human Sigmar1 colocalization assessed with mCherry tagged Sec16β protein and immunofluorescence staining of mitochondrial outer membrane protein Tom20 ii. GFP-tagged Sigmar1 fusion constructs | i. and ii. Exogenous overexpression of GFP-tagged fusion constructs | Zhemkov et al., 2021 |