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Western blot analysis of SMN on a HepG2 cell lysate (Human hepatocellular carcinoma; ATCC HB-8065) (left). Lane 1: 1:5000, lane 2: 1:10,000, lane 3: 1:20,000 dilution of the mouse anti-SMN antibody.
Immunohistochemical staining of pyrimidal cells in a rat cortex, formalin-fixed paraffin-embedded tissue section, with citrate pre-treatment (magnification, 20X) (center).
Immunofluorescent staining of differentiated SH-SY5Y cells (right). Cells were seeded in a 96 well, collagen coated imaging plate (Material # 353219) at ~ 5,000 cells per well. Cells were incubated with 50 mM ATRA (Sigma, R2625) for 5 days, followed by 50 ng/ml BDNF (Sigma, B3795) for 5 days. Differentiated cells were fixed and stained using the Triton X100 fix/perm protocol (see Recommended Assay Procedure; Bioimaging protocol link) and the anti-SMN antibody. The second step reagent was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen)(pseudo colored green). Cell nuclei were counter stained with Hoechst 33342 (pseudo colored blue). The image was taken on a BD Pathway™ 855 or 435 Bioimager System using a 20x objective and merged using the BD AttoVison ™ software This antibody also stained undifferentiated SH-SY5Y, SK-N-SH, C6, U87 and U373 cells using both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure; Bioimaging protocol link).
Western blot analysis of SMN on a HepG2 cell lysate (Human hepatocellular carcinoma; ATCC HB-8065) (left). Lane 1: 1:5000, lane 2: 1:10,000, lane 3: 1:20,000 dilution of the mouse anti-SMN antibody.
Immunohistochemical staining of pyrimidal cells in a rat cortex, formalin-fixed paraffin-embedded tissue section, with citrate pre-treatment (magnification, 20X) (center).
Immunofluorescent staining of differentiated SH-SY5Y cells (right). Cells were seeded in a 96 well, collagen coated imaging plate (Material # 353219) at ~ 5,000 cells per well. Cells were incubated with 50 mM ATRA (Sigma, R2625) for 5 days, followed by 50 ng/ml BDNF (Sigma, B3795) for 5 days. Differentiated cells were fixed and stained using the Triton X100 fix/perm protocol (see Recommended Assay Procedure; Bioimaging protocol link) and the anti-SMN antibody. The second step reagent was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen)(pseudo colored green). Cell nuclei were counter stained with Hoechst 33342 (pseudo colored blue). The image was taken on a BD Pathway™ 855 or 435 Bioimager System using a 20x objective and merged using the BD AttoVison ™ software This antibody also stained undifferentiated SH-SY5Y, SK-N-SH, C6, U87 and U373 cells using both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure; Bioimaging protocol link).
Western blot analysis of SMN on a HepG2 cell lysate (Human hepatocellular carcinoma; ATCC HB-8065) (left). Lane 1: 1:5000, lane 2: 1:10,000, lane 3: 1:20,000 dilution of the mouse anti-SMN antibody.
Immunohistochemical staining of pyrimidal cells in a rat cortex, formalin-fixed paraffin-embedded tissue section, with citrate pre-treatment (magnification, 20X) (center).
Immunofluorescent staining of differentiated SH-SY5Y cells (right). Cells were seeded in a 96 well, collagen coated imaging plate (Material # 353219) at ~ 5,000 cells per well. Cells were incubated with 50 mM ATRA (Sigma, R2625) for 5 days, followed by 50 ng/ml BDNF (Sigma, B3795) for 5 days. Differentiated cells were fixed and stained using the Triton X100 fix/perm protocol (see Recommended Assay Procedure; Bioimaging protocol link) and the anti-SMN antibody. The second step reagent was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen)(pseudo colored green). Cell nuclei were counter stained with Hoechst 33342 (pseudo colored blue). The image was taken on a BD Pathway™ 855 or 435 Bioimager System using a 20x objective and merged using the BD AttoVison ™ software This antibody also stained undifferentiated SH-SY5Y, SK-N-SH, C6, U87 and U373 cells using both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure; Bioimaging protocol link).
Western blot analysis of SMN on a HepG2 cell lysate (Human hepatocellular carcinoma; ATCC HB-8065) (left). Lane 1: 1:5000, lane 2: 1:10,000, lane 3: 1:20,000 dilution of the mouse anti-SMN antibody.
Immunohistochemical staining of pyrimidal cells in a rat cortex, formalin-fixed paraffin-embedded tissue section, with citrate pre-treatment (magnification, 20X) (center).
Immunofluorescent staining of differentiated SH-SY5Y cells (right). Cells were seeded in a 96 well, collagen coated imaging plate (Material # 353219) at ~ 5,000 cells per well. Cells were incubated with 50 mM ATRA (Sigma, R2625) for 5 days, followed by 50 ng/ml BDNF (Sigma, B3795) for 5 days. Differentiated cells were fixed and stained using the Triton X100 fix/perm protocol (see Recommended Assay Procedure; Bioimaging protocol link) and the anti-SMN antibody. The second step reagent was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen)(pseudo colored green). Cell nuclei were counter stained with Hoechst 33342 (pseudo colored blue). The image was taken on a BD Pathway™ 855 or 435 Bioimager System using a 20x objective and merged using the BD AttoVison ™ software This antibody also stained undifferentiated SH-SY5Y, SK-N-SH, C6, U87 and U373 cells using both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure; Bioimaging protocol link).
SMN (survival motor neuron) was discovered as a candidate gene, located in chromosome 5q13, for the fatal autosomal Spinal muscular atrophy (SMA) disorder. The SMN gene was missing or interrupted in a significant number of patients with SMA. The SMN protein is 294 amino acids and migrates with apparent molecular weight of 40 kDa. In addition to the cytoplasm, other studies localized SMN in dots of 0.1-1.0 µm within the nucleus. These novel nuclear structures were named "gems" and found associated to coiled bodies. It was also found that SMN interacts with the RGG box of hnRNP U and fibrillarin. Therefore, the biochemical function of SMN may be in the regulation of mRNA metabolism.
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