Delayed translocation of NGFI–B/RXR in glutamate stimulated neurons allows late protection by 9-cis retinoic acid

Calcium‐induced apoptosis of developing cerebellar granule neurons depends causally on NGFI‐B

“…Western analysis showed that NGFI‐B protein was present in cultures of rat cerebellar granule neurons at DIV 4/5 in concordance with former studies (Mathisen et al, 2011). The applied antibody targeting NGFI‐B had previously been validated (Mathisen et al, 2011; Strom and Paulsen, 2012) and a positive control was included to identify correct band size (results not included). Three siRNA sequences were tested, and the sequence specified in Section 2 was found to be effective.…”

“…These experiments were then excluded. We have previously confirmed that other apoptotic stimuli to cerebellar granule neurons cause translocation of NGFI‐B by using both immunostaining techniques (Jacobs et al, 2006b) and live cell imaging with gfp‐tagged proteins as was chosen in the present study (Mathisen et al, 2011). In these neuronal cultures 1 μM calcium ionophore increases expression and induces translocation of NGFI‐B (Boldingh Debernard et al, 2012).…”

“…In these neuronal cultures 1 μM calcium ionophore increases expression and induces translocation of NGFI‐B (Boldingh Debernard et al, 2012). However, as this dose was not suited for the present study, it was necessary to confirm that 0.1 μM calcium ionophore could recruit NGFI‐B similarly as seen after exposure to validated apoptotic stimuli, such as glutamate (Mathisen et al, 2011). Expression of NGFI‐B mRNA and cytosolic protein increased within 2 h after exposure to 0.1 μM calcium ionophore.…”

“…To enrich samples with cytosolic proteins and remove nuclear proteins, cells were harvested in 0.25 M sucrose with 100 mM Hepes pH 7.4, centrifuged (3500 rpm, 4 °C, 15 min) before SDS was added to a concentration of 2%. The efficiency of this protocol has been assessed previously (Mathisen et al, 2011). The following protease inhibitors were used: leupeptin (5 μg/μL), pepstatin A (1 μg/μL), PMSF (300 μM), and phosphatase inhibitor Na 3 VO 4 (100 μM) as NGFI‐B is a phosphoprotein.…”

“…Anti‐β‐actin (1:5000) was used for whole cell lysates. For samples enriched with cytosolic proteins anti‐α‐tubulin (1:1000) was chosen as we have used this antibody when validating the efficiency of protein enrichment in such samples previously (Mathisen et al, 2011). Immunoreactive proteins were visualized with a HRP substrate through the use of a gel doc system (Syngene, Cambridge, UK).…”