Fig 1: Validation of novel SPPL3 substrates in SPPL3 KO and SPPL3 D271A knock-in cells. a Immunoblot validation of the newly identified SPPL3 substrates CHST3, GALNT2 and MGAT1 in HEK293 cells. TCA-precipitated CM and membrane lysates obtained from three distinct SPPL3 KO clones and parental HEK293 cells (ctrl) were probed using specific antibodies. Calnexin served as loading control for the membrane fraction, total protein staining with Coomassie Brilliant Blue (CBB) as loading control for CM samples. b Immunoblot validation of the CHST3, GALNT2 and MGAT1 in HeLa cells. c Overview of SPPL3 topology (loops are not to scale). D271 targeted is highlighted in red, the GIGD motif in bold font. d Sanger sequencing reads confirming correct genome editing in SPPL3 D271A knock-in HEK293 cells. The targeted region of the SPPL3 locus was amplified by PCR from genomic DNA isolated from the indicated clones, gel-purified and Sanger sequenced. Reads were aligned to the corresponding chromosome 12 reference sequence. Encoded SPPL3 primary structure is provided for the correct reading frame. Mutated residues are highlighted by a red background. All clones analysed carry only the desired D271A missense mutation (red aa residue) in the active site of SPPL3. Additional mutations were introduced to prevent Cas9 re-cleavage, but remained silent (orange aa residues). e Phenotypic validation of SPPL3 D271A knock-in cells in comparison to SPPL3 KO and parental HEK293 cells. Secretion of B4GALT1 from cells was examined by immunoblotting of TCA-precipitated samples. Lysates of carbonate-washed membranes from the indicated cell clones were probed for the SPPL3 substrate MGAT5 and B4GALT1 as well as for SPPL3. f Immunoblot analysis of CHST3, GALNT2 and MGAT1 levels in TCA-precipitated supernatant and membrane lysates of isogenic SPPL3 D271A HEK293 cells
Fig 2: NGF-induced PC12 cell differentiation increases Golgi volume and transport proteins. (A,D,E) Immunofluorescence performed in PC12 cells differentiated with NGF (100 ng/ml) during the indicated days and stained with the specified markers. Nuclei were stained with Hoechst (blue). (A,C) The Golgi complex was labeled with GM130 (red, Bar: 8 μm) and the normalized fluorescence intensity was plotted (a total of 60 cells were analyzed). (B) Golgi volume was quantified on the indicated days. Fiji-ImageJ software was used to perform three-dimensional reconstruction and quantification of the images. (D,E) Rab1b, GM130, and GalNAc-T2 staining. Scale bars: 20 and 8 μm, respectively. (F) Representative Western blot assays performed with cell lysates obtained from PC12 cells differentiated with NGF during the indicated days (D2, D4, and D6). (G) Densitometric quantification of proteins shown in (F) normalized to β-actin. Values represent fold change relative to protein levels in untreated cells (D0). (H) Quantification of the indicated genes by qRT-PCR performed with total RNA during the indicated times. Results were normalized to the levels of TBP and expressed according to the 2–ΔΔCt method relative to the expression level of each gene in untreated cells (0 h, set as 1). Bars represent the mean ± SEM of three independent experiments carried out in triplicates. (B,C,G,H) Statistical data analysis were performed using ANOVA test, followed by Bonferroni multiple comparison post-test, considering statistically significant a value of p < 0.05 (∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001).
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