trim21 his protein  (Sino Biological)

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: E3 ubiquitin ligase TRIM21 mediates the degradation of HuR in response to heat shock (A) Immunoblot of lysates of MCF7 cells either unexposed or exposed to heat shock at 43°C in presence of 10% FBS and collected at the indicated time points, probed with HuR and β Actin antibodies. Quantification of relative HuR protein levels (normalized to β Actin) with HuR protein level in cells not exposed to heat shock taken as 1. Quantification of relative fold change in HuR mRNA levels of MCF7 cells exposed to heat shock for the indicated time points with HuR mRNA level in cells not exposed to heat shock taken as 1. GAPDH mRNA level was used to normalize HuR mRNA levels. The data represent mean ± SD values from three independent experiments. (B) Fluorescent confocal microscopic time-lapse images (60X) of live cells expressing HuR WT- EGFP fusion protein imaged for a period of 2 h at 37°C or 43°C. The scale bar is 20 μm. (C) Immunoblot of lysates of cells treated with CHX at 37°C or at 43°C or with both CHX and MG132 at 43°C and collected at the indicated time points, probed with antibodies against HuR and β Actin. (D) Quantification of relative HuR protein levels (normalized to β Actin) plotted against indicated time points for each experimental setup in (C). Data represent mean ± SD values from three independent experiments. (E) Immunoblot of lysates of cells transfected with either pCDNA3-HuR WT_Myc-His (HuR-WT) or pCDNA3-HuR-K182R_Myc-His (HuR-K182R) and exposed to heat shock for 2 h after 48 h of transfection. The blot was probed with His, HuR and β Actin antibodies. (F) Cells expressing HA-tagged Ubiquitin and His-tagged HuR-WT or HuR-K182R mutant proteins were treated with MG132 and subsequently exposed or unexposed to heat shock for 1 h. His-tagged proteins were purified from the cell lysate by Ni-NTA affinity pulldown and the precipitates probed with ubiquitin, His and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (G) Lysates of cells co-transfected with HuR-WT and TRIM21 expressing constructs, treated with MG132 and exposed to 37°C or 43°C, were immunoprecipitated with non-immune IgG or HuR or TRIM21 antibodies and the immunoprecipitates probed with TRIM21, HuR and GAPDH antibodies. Left panels represent the input lysates probed with the same antibodies. (H) Lysates of cells transfected with either control siRNA or TRIM21 siRNA and either exposed or unexposed to heat shock for 2 h, after 24 h of siRNA transfection, immunoblotted with TRIM21, HuR and β Actin antibodies. (I) Lysates of cells transfected with two increasing doses of TRIM21-expressing construct and either exposed or unexposed to heat shock for 2 h, after 48 h of transfection, immunoblotted with TRIM21, HuR and β Actin antibodies. (J) Lysates of cells co-transfected with TRIM21, Myc-His-tagged HuR-WT or HuR-K182R-expressing constructs and exposed to heat shock for 2 h, immunoblotted with TRIM21, Myc and β Actin antibodies. (K) Lysates of cells co-transfected with TRIM21 and His tagged HuR-WT or HuR-K182R-expressing constructs, treated with MG132 and exposed to 1 h of heat shock, were subjected to Ni-NTA affinity pulldown of His-tagged proteins. The precipitates were immunoblotted with TRIM21, His and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. ∗ signifies p value ≤0.05, ∗∗∗ signifies p value ≤0.005 (paired two-tailed t-test) in all panels. See also Figures S1–S5 .

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Transfection, Mutagenesis, Purification, Construct, Immunoprecipitation, Control, Two Tailed Test

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: TRIM21 protects cells from heat shock-induced cell death by degrading HuR (A) MCF7 cells transfected either with TRIM21-expressing plasmid or TRIM21 siRNA (siTRIM21) were unexposed or exposed to heat shock at 43°C for 2 h. MTT assay of cells was performed after 2 h of heat shock and optical density (OD) at 595 nm was measured. Data represent mean ± SD values from three independent experiments. (B) Cells transfected either with TRIM21-expressing plasmid or siTRIM21 were unexposed or exposed to heat shock at 43°C for 2 h. Caspase 3/7 activity assay with a luminescent substrate was performed immediately after 2 h of heat shock. Relative light unit (RLU) values of each sample are expressed as fold change from that of cells transfected either with empty vector or control siRNA at 37°C, taken as 1. Data represents mean ± SD values from three independent experiments. (C) Cells transfected either with empty vector (control) or TRIM21-expressing plasmid (TRIM21 OE) were unexposed or exposed to heat shock at 43°C for 2 h, after 48 h of transfection. Cells were then stained with AnnexinV-AlexaFluor 488 and PI to detect apoptosis by fluorescent microscopy. Quantification of fluorescent intensity per cell of Annexin V and PI staining was determined and is depicted in the boxplots in the right panel. Data represent mean ± SD values of fluorescent intensity/cell from 100 cells that were randomly selected from each treatment. Scale bars are 100 μm. (D) Cells transfected either with control siRNA or siTRIM21 were unexposed or exposed to heat shock at 43°C for 2 h after 48 h of transfection. Cells were then processed and data represented as in (C). (E) Cells transfected either with empty vector (control) or TRIM21-expressing plasmid (TRIM21 OE) were unexposed or exposed to heat shock at 43°C for 2 h after 48 h of transfection. Cells were then imaged at 10X in phase contrast microscope and projected cell areas and cell perimeters were determined using ImageJ software. Data represent mean ± SD values from 400 cells randomly selected from each experimental setup in individual biological repeats. (F) Cells transfected either with control siRNA or siTRIM21 were unexposed or exposed to heat shock at 43°C for 2 h after 48 h of transfection. Data was then collected and analyzed as in (E). (G) Cells transfected either with empty vector (control) or TRIM21-expressing plasmid (TRIM21 OE) were unexposed or exposed to heat shock at 43°C for 2 h after 48 h of transfection, following which they were allowed to grow at 37°C for 72 h. Cells were collected at indicated time points and MTT assay was performed. Data represent mean ± SD values from three independent experiments. (H) Cells transfected either with control siRNA or siTRIM21 were unexposed or exposed to heat shock at 43°C for 2 h after 48 h of transfection, following which they were allowed to grow at 37°C for 72 h. Cells were collected at indicated time points and MTT assay was performed. Data represent mean ± SD values from three independent experiments. (I) MCF7 cells transfected either with empty vector (control) or TRIM21-expressing plasmid (TRIM21 OE) were unexposed or exposed to heat shock at 43°C for 2 h after 48 h of transfection, following which 10 3 cells were seeded and allowed to form colonies at 37°C. After 14 days, colonies were counted by crystal violet staining (left panel). Relative colony forming unit (CFU) values from 3 independent experiments were plotted (right panel). (J) MCF7 cells transfected with either empty vector (Mock), HuR WT, HuR K182R and TRIM21-expressing plasmids, individually or in combination, were either unexposed or exposed to heat shock at 43°C for 2 h. MTT assay was performed immediately after 2 h of heat shock (left panel). Caspase 3/7 activity assay with a luminescent substrate was performed immediately after 2 h of heat shock. RLU values of each sample are expressed as fold change from that of mock-transfected cells at 37°C, taken as 1 (right panel). Data for both panels represent mean ± SD values from three independent experiments. (K) MCF7 cells transfected with either empty vector (Mock), HuR WT, HuR K182R and TRIM21-expressing plasmids, individually or in combination, were either unexposed or exposed to heat shock at 43°C for 2 h following which they were allowed to grow at 37°C for 72 h. MTT assay was performed at the indicated time points. Data represent mean ± SD values from three independent experiments. ∗, #, $, @, α, β, γ signifies p value ≤0.05, ∗∗, ##, $$, @@, αα, ββ, γγ signifies p value ≤0.01 and ∗∗∗, ###, $$$, @@@, ααα, βββ, γγγ signify p value ≤0.005 (paired two-tailed t-test) in all panels. See also Figures S6–S8 .

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Transfection, Expressing, Plasmid Preparation, MTT Assay, Activity Assay, Control, Staining, Microscopy, Software, Two Tailed Test

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: S100 and E101 residues of HuR are determinants of recognition by TRIM21 (A) Schematic representation of HuR WT and deletion constructs, indicating the 3 RNA recognition motifs (RRMs), nuclear localization signal (NLS), linker and hinge regions of HuR. (B) Immunoblot of lysates of cells expressing His-tagged HuR WT and various His-tagged deletion mutants either exposed or unexposed to heat shock at 43°C for 2 h, probed with antibodies against His tag and β Actin. (C) Cells co-expressing TRIM21 with either His-tagged HuR WT or various deletion mutants were exposed to heat shock at 43°C for 1 h in presence of MG132. Lysates were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies with HuR antibody. (D) Contact map of HuR residues with TRIM21 counterparts generated by MD simulation, contact being defined as a distance less than 10 Å between respective α-carbons of amino acids. (E) Structure of HuR RRM1-2 (PDB: 4EGL ) showing the molecular distance between S100 and K182 and between R115 and K182 residues. (F) Still from the MD simulation showing the bound state of RRM1-2 domain of HuR (blue) with the PRYSPRY domain of TRIM21 (red). The zoomed-in image shows the close contact between S100 of HuR (licorice structure) and the PRYSPRY domain of TRIM21. See also Figures S9 and .

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Construct, Western Blot, Expressing, Generated

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: S100 and E101 residues of HuR determine TRIM21 binding and degradation of HuR under heat shock (A) Schematic representation of HuR WT and point mutants indicating the point mutations S100A, E101A, S100A/E101A double mutant and R115A/T116A double mutant. (B) Immunoblot of lysates of MCF7 cells expressing either His-tagged HuR WT or HuR E101A or HuR S100A or HuR S100A/E101A mutant exposed or unexposed to heat shock at 43°C for 2 h, probed with antibodies against His tag and β Actin. (C) Lysates of cells expressing TRIM21 together with either His-tagged HuR WT or HuR E101A or HuR S100A or HuR S101A/E101A mutant proteins, exposed to heat shock at 43°C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (D) Lysates of cells expressing HA-tagged Ubiquitin together with either His-tagged HuR WT or HuR S100A/E101A mutant proteins, exposed to heat shock at 43°C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with Ubiquitin, His, and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. Ubiquitinated protein band intensities, normalized to that from HuR WT expression, in absence of heat shock, are represented below the ubiquitin immunoblot. (E) MCF7 cells transfected with either empty vector (Mock), HuR WT, HuR S100A/E101A and TRIM21 expression constructs individually or in combination were unexposed or exposed to heat shock at 43°C for 2 h. MTT assay was performed immediately after 2 h of heat shock (top left panel). Caspase 3/7 activity assay was performed with MCF7 cells transfected with the same combinations of expression constructs and unexposed or exposed to heat shock at 43°C for 2 h. RLU values of each sample are expressed as fold change from that of mock-transfected cells at 37°C, taken as 1 (bottom left panel). Data represent mean ± SD values from three independent experiments for both panels. MCF7 cells transfected with the same combinations of plasmid DNA and exposed or not exposed to heat shock for 2 h, were stained with AnnexinV-AlexaFluor488 and PI to detect apoptosis. Fluorescence intensity of the Annexin V (top right panel) and PI stained (bottom right panel) cells was measured. Data represent mean ± SEM values from 100 cells randomly selected from each experimental setup. (F) Cells transfected with either empty vector (Mock), HuR WT, HuR S100A/E101A and TRIM21 expression constructs individually or in combination were unexposed (upper panel) or exposed (lower panel) to heat shock at 43°C for 2 h, following which they were allowed to grow at 37°C for 72 h. MTT assay was performed at the indicated time points. Data represent mean ± SD values from three independent experiments. (G) Cells were transfected with HuR WT or HuR S100A/E101A expressing constructs and exposed or not exposed to heat shock at 43°C for 2 h. mRNA expression levels of HuR targets were determined by quantitative RT-PCR using target specific primers from total RNA isolated from these cells. GAPDH mRNA was used as normalization control. The data represents fold change in target mRNA transcripts from that HuR WT-expressing cells at 37°C, taken as 1. Data represents mean ± SD values from three independent experiments. Immunoblots using His and β Actin antibodies of lysates of cells transfected with HuR WT or HuR S100A/E101A expression constructs and exposed or not exposed to heat shock from which RNA was isolated for qRT-PCR (lower panel). ∗, #, $, @ signifies p value ≤0.05, ∗∗, ##, $$, @@ signifies p value ≤0.01 and ∗∗∗, ###, $$$, @@@ signifies p value ≤0.005 (paired two-tailed t-test) in all panels. See also Figures S11–S15 .

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Binding Assay, Mutagenesis, Western Blot, Expressing, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Construct, MTT Assay, Activity Assay, Staining, Fluorescence, Quantitative RT-PCR, Isolation, Control, Two Tailed Test

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: S100 phosphorylation is required for TRIM21 binding and degradation of HuR under heat shock (A) Lysates of MCF7 cells expressing His-tagged HuR WT or HuR S100A mutant proteins, exposed to heat shock at 43°C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with phosphoserine, His and β Actin antibodies. Left panels represent the input lysates probed with His and β Actin antibodies. (B) Immunoblot of lysates of cells expressing His-tagged HuR WT or HuR S100D mutant proteins exposed or not exposed to heat shock at 43°C for 2 h and probed with antibodies against His tag and β Actin. (C) Immunoblot of lysates of cells transfected with control siRNA or siTRIM21 together with His-tagged HuR S100D expression construct and not exposed to heat shock, probed with antibodies against TRIM21, His tag and β Actin. (D) Lysates of cells expressing TRIM21 and His-tagged HuR WT or HuR S100D mutant proteins, exposed to heat shock at 43°C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (E) Cells transfected with either empty vector (Mock), HuR WT or HuR S100D expression constructs were not exposed or exposed to heat shock at 43°C for 2 h. MTT assay was performed immediately after 2 h of heat shock. Data represent mean ± SD values from three independent experiments. (F) Cells transfected with either empty vector (Mock), HuR WT or HuR S100D expression constructs were unexposed or exposed to heat shock at 43°C for 2 h. Caspase 3/7 activity assay was performed. Relative light unit values of each sample, representing caspase 3/7 activity, are expressed as fold change from that of mock transfected cells at 37°C, taken as 1. Data represent mean ± SD values from three independent experiments. ∗, #, $ signifies p value ≤0.05, ∗∗, ##, $$ signifies p value ≤0.01 and ∗∗∗, ###, $$$ signifies p value ≤0.005 (paired two-tailed t-test) in all panels. See also Figure S16 .

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Mutagenesis, Western Blot, Transfection, Control, Construct, Plasmid Preparation, MTT Assay, Activity Assay, Two Tailed Test

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: AKT1 phosphorylates S100 leading to TRIM21-mediated degradation of HuR under heat shock (A) Phylogenetic analysis of HuR sequences from vertebrate species showing evolutionary conservation of AKT1 target consensus motif (RXXS/T) at S100. (B) Immunoblot of lysates of MCF7 cells treated with DMSO or 10 μM Akt1 inhibitor for 2 h and subsequently unexposed or exposed to heat shock at 43°C for 2 h, probed with antibodies against HuR and β Actin. (C) Immunoblot of lysates of cells transfected with control siRNA or Akt1 siRNA, and unexposed or exposed to heat shock at 43°C for 2 h, after 48 h of siRNA transfection, probed with antibodies against AKT1, HuR and β Actin. (D) Immunoblot of lysates of cells exposed to heat shock at 43°C for the indicated time durations, probed with antibodies against phosphoAKT1, AKT1, HuR and β Actin. (E) Lysates of cells treated with MG132 for 1 h and unexposed or exposed to heat shock at 43°C for 2 h, were immunoprecipitated with non-immune IgG and HuR antibody and the immunoprecipitates were probed with AKT1, phosphoAKT1, HuR and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (F) Cells were treated with DMSO or 10 μM Akt inhibitor for 2 h followed by 1 h MG132 treatment, and unexposed or exposed to heat shock at 43°C for 2 h. Cell lysates were immunoprecipitated with non-immune IgG and HuR antibody and the immunoprecipitates were probed with phosphoserine, HuR and β Actin antibodies. Left panels represent the input lysates probed with HuR and β Actin antibodies. (G) Lysates of cells treated with DMSO or 10 μM Akt inhibitor for 2 h and MG132 for 1 h and unexposed or exposed to heat shock at 43°C for 2 h, were immunoprecipitated with non-immune IgG and HuR antibody and the immunoprecipitates were probed with phosphoAKT1, HuR and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (H) Lysates of cells expressing His-tagged HuR WT or HuR S100A and treated with MG132 and unexposed or exposed to heat shock at 43°C for 1 h, were subjected to Ni-NTA affinity pulldown. The precipitates were probed with antibodies against phosphoAKT1, His and β Actin. Left panels represent the input lysates probed with the same antibodies. (I) Immunoblot of lysates from cells expressing His-tagged HuR WT or HuR R97A mutant proteins, not exposed or exposed to shock at 43°C for 2 h, probed with antibodies against His tag and β Actin. (J) Lysates of cells expressing His-tagged HuR WT or HuR R97A and treated with MG132 and unexposed or exposed to heat shock at 43°C for 1 h, were subjected to Ni-NTA affinity pulldown. The precipitates were probed with antibodies against phosphoserine, His and β Actin. Left panels represent the input lysates probed with the same antibodies. (K) Lysates of cells expressing His-tagged HuR WT or HuR R97A and treated with MG132 and not exposed or exposed to heat shock at 43°C for 1 h, were subjected to Ni-NTA affinity pulldown. The precipitates were probed with antibodies against phosphoAKT1, His and β Actin. Left panels represent the input lysates probed with the same antibodies. (L) In vitro serine phosphorylation of HuR by AKT1. Purified AKT1 enzyme was first incubated with 1 μg of MCF7 cell lysate from cells either transfected with control siRNA or Akt1 siRNA to deplete the endogenous AKT1 protein and exposed to either 37°C or heat shock at 43°C. Purified, His-tagged HuR protein was incubated with the activated AKT1 protein in presence of ATP as phosphate donor, and then purified using Ni-NTA chromatography and probed with phosphoserine and His antibodies. (M) Purified AKT1 enzyme was first incubated with 1 μg of MCF7 cell lysate from cells either transfected with control siRNA or Akt1 siRNA to deplete the endogenous AKT1 protein and exposed to either 37°C or heat shock at 43°C in absence or presence of 10 μM Akt inhibitor. Purified, His-tagged HuR protein was incubated with the AKT1 protein in presence of ATP as phosphate donor, and then purified using Ni-NTA chromatography and probed with phosphoserine and His antibodies. (N) Purified AKT1 enzyme was first incubated with 1 μg of MCF7 cell lysate from cells either transfected with control siRNA or Akt1 siRNA to deplete the endogenous AKT1 protein and exposed to either 37°C or heat shock at 43°C. Purified, His-tagged WT HuR or HuR S100A protein was incubated with the activated AKT1 protein in presence of ATP as phosphate donor, and then purified using Ni-NTA chromatography and probed with phosphoserine and His antibodies. See also Figures S17–S19 .

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Western Blot, Transfection, Control, Immunoprecipitation, Expressing, Mutagenesis, In Vitro, Phospho-proteomics, Purification, Incubation, Chromatography

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: Concerted action by AKT1 and TRIM21 causes HuR degradation under heat shock (A) Cells were co-transfected with TRIM21 and His-tagged HuR WT expression constructs, treated with 10 μM Akt Inhibitor for 2 h followed by 1 h MG132 treatment, and unexposed or exposed to heat shock at 43°C for 2 h. Cell lysates were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and β Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (B) Cells were co-transfected with TRIM21 and His-tagged HuR WT expression constructs along with control siRNA or Akt1 siRNA. After 46 h of transfection and 1 h of MG132 treatment, cells were unexposed or exposed to heat shock at 43°C for 1 h. Lysates were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His, β Actin and AKT1 antibodies. Left panels represent the input lysates probed with the same antibodies. (C) Lysates of cells expressing TRIM21 together with His-tagged HuR WT or HuR R97A proteins and treated with MG132 and unexposed or exposed to heat shock at 43°C for 1 h, were subjected to Ni-NTA affinity pulldown. The precipitates were probed with antibodies against TRIM21, His and β Actin. Left panels represent the input lysates probed with the same antibodies. (D) In vitro ubiquitination of p -Ser HuR by TRIM21. Purified His-tagged HuR was either untreated or treated with purified AKT1 protein to cause serine phosphorylation. The serine-phosphorylated HuR was further purified using Ni-affinity chromatography and used as the substrate in in vitro ubiquitination reactions containing purified TRIM21, purified ubiquitin and 1 μg lysates of cells transfected with Ctrl. siRNA or TRIM21 siRNA and either exposed to 37°C or 43°C for 2 h. After the ubiquitination reaction, HuR was again purified by Ni-affinity chromatography and resolved by SDS-PAGE. Immunoblotting was performed using ubiquitin, phosphoserine and His antibodies to detect ubiquitinated, serine-phosphorylated and His-tagged HuR. (E) Proposed model showing the sequential actions of AKT1 and TRIM21 under heat shock, leading to phosphorylation of HuR on S100 and ubiquitination on K182, generating a heat-shock induced “phosphodegron” in HuR. This leads to the proteasomal degradation of HuR allowing cell survival and adaptation to heat shock. See also Figures S20–S22 .

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Transfection, Expressing, Construct, Control, In Vitro, Ubiquitin Proteomics, Purification, Phospho-proteomics, Affinity Chromatography, SDS Page, Western Blot

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet:

Article Snippet: His tagged TRIM21 protein , ProSpec Bio, New Brunswick, NJ , Cat # pro-328-a.

Techniques: Ubiquitin Proteomics, Purification, Virus, Recombinant, Transfection, Negative Control, Mutagenesis, Software, Microscopy