Fig 1: One-step generation of multi-gene knockouts in HeLa cells. (A) Design of the sgRNA targeting the consensus sequence of five HSPA family genes and the universal linear donor (DonorHSPA) used for the enrichment of cells containing multi-gene mutations. The consensus sequence of the HSPA gene family was analysed by multiple sequences alignment. The black-shaded nucleotides represent the consensus sequence of all five HSPA genes. The green-shaded nucleotides represent the consensus sequence of three or four HSPA genes, and the yellow-shaded nucleotides represent the nonconsensus nucleotides. (B) sgRNAHSPA-triggered indels on five target genes in the absence and presence of DonorHSPA, after puromycin selection. Indel efficiencies were evaluated with a T7E1 assay. Error bars indicate SD (n = 3), t-test, **P < 0.01, ***P < 0.001. (C) Partial coding sequences of HSPA1A,HSPA1B,HSPA1L and HSPA6 genes in the genome of HeLa clone 3 containing the sgRNA-targeting regions (red) and sequencing analysis of the mutated alleles. Clone 3 was from HeLaOC cells transfected with sgRNAHSPA/DonorHSPA. Shaded nucleotides represent the PAM sequence, and the dashes indicate deletions. Light-grey arrows in the background indicate the CMV promoter direction in the donor. (D) mRNA levels of HSPA1A and HSPA1B (normalized to GAPDH) of clone 3 was quantified by real-time PCR analysis. (E) Western blotting analysis for HSPA1L and ß-tubulin (loading control) of clone 3.
Fig 2: SteE Interacts with Catalytically Active GSK3 and STAT3(A) Whole-cell lysates from 293ET cells expressing GFP or GFP-SteE were analyzed by immunoblot with antibodies against STAT3, pY705-STAT3, GFP, and tubulin (Tub) as a loading control. Data are representative of three independent experiments.(B) GFP or GFP-SteE was expressed in 293ET cells, immunoprecipitated, and assessed for its ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay. The * represents endogenous phosphorylated STAT3 that was detected upon immunoprecipitation with GFP-SteE but not with GFP. Data are representative of three independent repeats.(C) pBMDMs were infected with steE mutant Salmonella carrying pWSK29-SteE:HA or sseL mutant Salmonella carrying pWSK29-SseL:HA. 17 h after uptake, HA-tagged effectors were immunoprecipitated from cell lysates and assessed for their ability to bind endogenous GSK3, STAT3, STAT6, or HSPA1L as indicated. Immunoblots are representative of three independent experiments.(D) GFP or GFP-SteE, expressed in 293ET cells, was immunoprecipitated and assessed for its ability to interact with the indicated co-expressed FLAG-tagged GSK3 variants and endogenous GSK3 (indicated with arrows). Data are representative of three experiments. The * indicates a higher-molecular-weight form of SteE.
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