rabbit anti-als2  (Millipore)

Journal: eLife

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.7554/elife.32282

Figure Lengend Snippet: Figure 10. Localization of Rabex-5 and Alsin upon mitochondrial stress. BAC GFP-Rab5 cells seeded on gridded dishes were labeled with 100 nM Mito-Red and photoirradiated as before. Cells were fixed after 30 min post-laser treatment and immunostained with antibody against Rabex-5 (A) or Alsin (B). Inset regions are shown. Scale bars, 10 mm. (C) Colocalization analysis from untreated and laser-treated cells in (A) and (B), n = 3. p-Values based on two-tailed t-tests. DOI: https://doi.org/10.7554/eLife.32282.042 The following source data and figure supplements are available for figure 10:

Article Snippet: Cells were immunostained with the corresponding primary antibodies: anti-rabbit Rabenosyn-5/ ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse APPL2 (home-made), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Labeling, Two Tailed Test

Journal: eLife

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.7554/elife.32282

Figure Lengend Snippet: Figure 11. Alsin is required for Rab5 recruitment and regulates cytochrome c release. (A) Flow chart depicting the different stages and time (in days) from induced-pluripotent stem cells (iPSC), to neuroprogenitor cells (NPC), and to mature spinal motor neurons (sMN). The small molecules and compounds used at different stages are shown and color-coded. (B) WT and Alsin-/- cells were challenged with either PBS (Ctrl) or 100 mM H2O2 at 37˚C for 1 hr. Cells were fixed and immunostained with Rab5 and TOM20 antibodies. Inset images show are shown. Scale bars, 10 mm. (C) Subcellular fractionation of mitochondrial (Mito) and cytosolic (C) fractions from WT and Alsin-/- iPSC- sMN challenged with either PBS or 100 mM H2O2 at 37˚C for 1 hr. Protein samples were loaded onto SDS-PAGE and imunoblotted with antibodies against Rab5, tubulin and TOM20. (D) Cytosolic fractions were prepared from WT and Alsin-/- iPSC-sMN challenged with 100 mM H2O2 at 37˚C for 1 hr. Densitometric quantification of cytosolic cytochrome c were collected from three independent experiments. Y-axis corresponds to normalized ratio intensity of cytochrome c to tubulin. Error bars represent SEMs. p-Values based on two-tailed t-tests. DOI: https://doi.org/10.7554/eLife.32282.047 The following source data and figure supplement are available for figure 11:

Article Snippet: Cells were immunostained with the corresponding primary antibodies: anti-rabbit Rabenosyn-5/ ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse APPL2 (home-made), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Fractionation, SDS Page, Two Tailed Test

Journal: eLife

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.7554/elife.32282

Figure Lengend Snippet: Figure 12. Schematic model depicting the role of Rab5-Alsin-mitochondria during oxidative stress. In the normal condition, mitochondria (Mito, red) are elongated and tubular (left). Rab5 (green) are localized on early endosomes (EE) to assemble the Rab5 machinery for endosomal maturation and membrane trafficking. At steady state, some EE make transient contacts with mitochondria. During oxidative stress (e.g. laser- or chemically- induced), mitochondria undergo MOMP and a dramatic morphological transformation into rounded and swollen structures (right). The release of the apoptotic factor, cytochrome c, from mitochondria into the cytosol is associated with a re-localization event of Rab5 from EE to mitochondria via a cytosolic intermediate, accompanied by an increase in EE-mitochondria MCS. The recruitment and activation of Rab5 on mitochondria depend on the Rab5 GEF Alsin (blue), which leads to a selective recruitment of Rabenosyn-5 (light green). This signaling cascade on mitochondria is a reversible process that regulates the apoptotic program (e.g. cytochrome c release and caspase activation) and thus, promotes overall cell survival. DOI: https://doi.org/10.7554/eLife.32282.050

Article Snippet: Cells were immunostained with the corresponding primary antibodies: anti-rabbit Rabenosyn-5/ ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse APPL2 (home-made), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Membrane, Transformation Assay, Activation Assay