Fig. 2

Identification of microglial translatome and LFQ quantification using MaxQuant.A Schematic representation of the ribosome affinity purification method from CD11br mice brain cortical tissue. B cDNAs synthesized from the purified RNA after immunoprecipitation of cortical lysates from WT and APPPS1-21-CD11br treated with vehicle or ABX was used for RT-PCR analysis. An amplicon corresponding to EGFP in the FLAG/EGFP-Rpl10a transgene mRNA was detected (a). No band was detected in APPPS1-21 and non-Tg samples that were used as controls for pull-down assay. Gapdh was used as reference gene. (b) qPCR analysis with the same primer set showed that the levels of EGFP were comparable between all the mice groups. 2^{Δ ΔCT} method was used to show the expression of FLAGEGFP-Rpl10a transgene wherein the difference between C_T values of the gene interest and reference gene is calculated for all the samples and plotted as a bar graph. The error bar indicates the SD of two technical replicates. UD refers to undetermined. C RT-PCR analysis of cDNAs from WT-CD11br male and female mice indicate amplification of microglia/macrophage-specific markers Iba1, P2ry12, Tmem119, Cx3cr1and Trem2. Gfap, Tubb3, Cldn11 were used as markers for astrocytes, neurons, and oligodendrocytes respectively. D Six-cohorts of mice including vehicle-treated WT-CD11br male (RM); CD11br female (RF); vehicle-treated APPPS1-21-CD11br male (TMV); vehicle-treated APPPS1-21-CD11br female (TFV); ABX-treated APPPS1-21-CD11br male (TMA); and ABX-treated APPPS1-21-CD11br female (TFA) mice were employed for proteomic analysis of the newly-synthesized peptides in microglia. Animals were sacrificed at 7 weeks of age (total n = 15, per group) and 3 half cortices from 3 mice were pooled into each sample based on their amyloid burden for each cohort (n = 5). Western blot after immunoprecipitation of cortical lysates from WT-CD11br mice with anti-flag antibody demonstrates the immunobinding of the F/EGFP-Rpl10a transgene to FLAG beads (b). F Principal component analysis (PCA) plot based on normalized data of the proteins identified from all six groups of the proteomic analysis. G Venn diagram representing the number of proteins found in all replicates of each group of male mice (RM, TMV, TMA) and female mice (RF, TFV, TFA). H Number of differentially expressed proteins (DEPs) for each comparison, black-total number of significant DEPs (Limma q-value < 0.05 and |z|> 1.96); red and blue indicate upregulated and downregulated proteins respectively