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. 2022 Nov 25;11(23):3770. doi: 10.3390/cells11233770

Figure 5.

Figure 5

UBA52 interacts with HSP90 and CHIP. Co-IP (Protein A Sepharose beads attached to the anti-UBA52 antibody/anti-IgG antibody) was performed, and the cell or tissue lysates were incubated with the antibody-beads complex, following which the beads were boiled with 2X-reducing buffer, centrifuged and the supernatant was resolved on a 4–16% gradient SDS-PAGE. The gel was stained with Coomassie, followed by distaining and band excision to identify the interacting proteins through mass-spectrometric-based detection. MS/MS peaks representing the fragmentation spectrum (highest intensity) obtained from the tryptic peptides of amino acids of HSP90 α/β in (a) SH-SY5Y cells and (b,c) in rat brain regions; nexp = 3. (d) The pie diagram illustrates the key chaperones obtained from the mass-spectrometric analysis. (e) Immunoblots representing the endogenous UBA52 and HSP90 with or without rotenone and MG132 treatment in SH-SY5Y cells; nexp = 3 and (f) in control and rotenone-lesioned SN and STR rat brain region; nexp = 4 after the Co-IP was performed using Protein A Sepharose beads and anti-UBA52 antibody/anti-IgG antibody complex incubated with cell and tissue lysates. (g) Next, Co-IP (Protein A Sepharose beads attached to the anti-UBA52 antibody/anti-IgG antibody) was utilized to detect the interaction of endogenous UBA52 and E3 ubiquitin ligase CHIP in SH-SY5Y cells with or without rotenone treatment with MG132, along with input bands nexp = 3. (h) Immunoblots representing the interaction of endogenous UBA52 and CHIP in control and rotenone-treated rat brain regions, substantia nigra (SN) and striata (STR) nexp = 3 after Co-IP with an anti-UBA52 antibody/anti-IgG antibody complexed with Protein A Sepharose beads. (i) Further, the SY-SY5Y cells were transiently overexpressed with Myc-α-synuclein (α-syn) to induce a pathological state. Next, the interaction of endogenous UBA52 with CHIP and UBA52 with HSP90 was assessed through immunoblotting after the Co-IP was performed using the complex of Protein A Sepharose beads and anti-UBA52 antibody/anti-IgG antibody incubated with PcDNA3.1 or Myc-tagged α-syn transfected SH-SY5Y cells after MG132 treatment; nexp = 3.