Protein concentration assay

Hippocampal slices were prepared from age-matched male WT and Fmr1 KO rats in an interleaved fashion as previously described (17 (link), 26 (link), 42 (link)). Briefly, hippocampi were rapidly isolated, and 500-μm slices were prepared from the dorsal half using a Stoelting tissue slicer. Slices were recovered for 4 hours in 32.5°C ACSF (NaCl, 124 mM; KCl, 3 mM; NaH₂PO₄, 1.25 mM; NaHCO₃, 26 mM; dextrose, 10 mM; MgCl₂, 1 mM; and CaCl₂, 2 mM, saturated with 95% O₂ and 5% CO₂) and then incubated for 30 min with 25 μM ActD to block transcription. To measure protein synthesis, slices were then transferred to fresh ACSF containing ³⁵S-Met/Cys (10 μCi/ml; PerkinElmer) and incubated for 30 min. After labeling, slices were homogenized, and labeled proteins were isolated by trichloroacetic acid precipitation. Samples were read with a scintillation counter and subjected to a protein concentration assay (Bio-Rad). Final data were expressed as counts per minute per microgram of protein, normalized to the ³⁵S-Met/Cys ACSF used for incubation, and the average incorporation of all samples was analyzed in that experiment (eight samples per experiment: four WT and four Fmr1 KO). All aspects of the experiments were performed blinded to genotype.

Whole brain lysates were harvested using 20 mM HEPES, 320 mM sucrose with protease inhibitors (0.1 µg/µl Leupeptin/Aprotinin and 1 mM PMSF). Protein concentration of lysate was determined using the Bio-Rad protein concentration assay and equal amounts of protein were loaded and subjected to SDS-PAGE gel electrophoresis followed by transfer to PVDF membranes (Immobilon-P). Membranes were probed with mouse anti-Kirrel3 1:100 (Neuromab Clone N321C/49; catalog no. 75–333) that detects the long and short isoforms of Kirrel3, rat anti-HSC70/HSP73 1:10,000 (Enzo Life Sciences), or rabbit anti-β-actin 1:1,000 (Cell Signaling Technology), followed with appropriate HRP-conjugated secondary antibodies. Blots were developed using SuperSignal West Femto Kit (Thermo Fisher Scientific). Quantification of relative protein levels was performed on scanned immunoblots using Fiji software.

Cells were lysed in M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) with protease and phosphatase inhibitors (Roche), and proteins were quantified using a protein concentration assay (Bio-Rad Laboratories). One microgram of the appropriate control immunoglobulin G, together with 20 μl of resuspended protein A/G PLUS-Agarose, was added to precleared lysate and incubated at 4°C for 30 min. Pellet beads were centrifuged at 2500 rpm for 5 min at 4°C, and the supernatant with total cellular protein (300 μg) was transferred to a fresh centrifuge tube on ice. Primary antibody was added and incubated overnight at 4°C. Twenty microliters of resuspended protein A/G PLUS-Agarose was added at 4°C. After 2 hours, the tubes were centrifuged at 2500 rpm for 5 min at 4°C and the immunoprecipitates were collected. The beads were pelleted and washed with radioimmunoprecipitation assay buffer. Beads were then pelleted, washed, and resuspended in 40 μl of electrophoresis sample buffer. The samples were boiled at 90°C for 5 min, and 20-μl aliquots were subjected to Western blotting analysis.