Journal: PLoS Pathogens

Article Title: The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid

doi: 10.1371/journal.ppat.1005833

Figure Lengend Snippet: (A) A3-RNA cross-linked complexes were immunoprecipitated from mock infected or HIV-1 NL4-3 ΔVif infected HEK 293T cells stably expressing 3×HA-tagged A3F, A3G or A3H proteins that had been fed with 4SU and UV-irradiated. A3-RNA cross-linked complexes were also immunoprecipitated from purified HIV-1 NL4-3 ΔVif virions that were derived from the 4SU-fed infected cells. Complexes were visualized by autoradiography (top) and Western blot analysis using a polyclonal anti-HA (bottom). (B) Frequency distribution of nucleotide occurrence (read density) in reads that were mapped to the HIV-1 NL4-3 genome (left). A colinear schematic diagram of the HIV-1 genome is presented above. Correlation analyses (see ) of A3 binding to viral RNA in cells from two independent CLIP experiments are shown (right).

Article Snippet: The rabbit polyclonal anti-HA (600-401-384 Rockland) and the mouse monoclonal anti-HIV-1 p24CA (183-H12-5C, NIH AIDS Reagent Program) were used in Western blot analyses.

Techniques: Immunoprecipitation, Infection, Stable Transfection, Expressing, Irradiation, Purification, Derivative Assay, Autoradiography, Western Blot, Binding Assay

Journal: Horticulture Research

Article Title: The key cyclic electron flow protein PGR5 associates with cytochrome b 6 f , and its function is partially influenced by the LHCII state transition

doi: 10.1038/s41438-021-00460-y

Figure Lengend Snippet: a CsPGR5a-HA , CsPetC , CsLhcb3 , and CsPsaH were coexpressed in N. benthamiana , and CoIP was performed by using an anti-HA antibody. Tobacco thylakoid membranes coexpressing empty vectors together with HA , CsPetC , CsLhcb3 , and CsPsaH were used as negative controls, and purified thylakoid membranes (Input) were used as positive controls. Western blotting was performed by using anti-PsaH, anti-PetC, and anti-Lhcb3 antibodies. b Immunoblotting analysis of cucumber thylakoid membrane protein complexes by using anti-PGR5 and anti-PetC antibodies. c BN-PAGE of tobacco thylakoid membrane proteins with or without overexpression of CsPGR5a , anti-PGR5, anti-PetC, anti-Lhcb3, and anti-PsaH antibodies was used for immunoblotting

Article Snippet: The thylakoid membranes were purified after 3 days and solubilized in TBS consisting of 1.5% (w/v) DM at a final concentration of 1 mg Chl·ml −1 for 30 min. After centrifugation at 12,000 × g , the supernatant and 70 μl of anti-HA magnetic beads (MCE, China) (which had been preequilibrated with TBS consisting of 0.08% [w/v] DM) were incubated together and shaken overnight at 4 °C.

Techniques: Purification, Western Blot, Membrane, Over Expression

Journal: Journal of Cancer

Article Title: N6-methyladenosine regulates ATM expression and downstream signaling.

doi: 10.7150/jca.64061

Figure Lengend Snippet: Figure 5. YTHDFs suppress ATM expression in the post-transcriptional levels. (A) SGC7901 cells were transfected with Flag-NC or Flag-YTHDF1 or Flag-YTHDF2 for 36 h, followed by CHX treatment (25 μg/ml) for indicated times. Cellular proteins were collected and Western blot was performed. (B) HEK293 cells were transfected with indicated plasmids (Flag-NC, Flag-YTHDF1 and HA-Ubiquitin), and cells were treated with MG132 (25 μM) for 4 h before harvest. Lysates were immunoprecipitated with anti-HA antibodies and then immunoblotted with anti-ATM antibodies. (C) HEK293 cells were transfected with Flag-NC or Flag-YTHDF1 for 48 h, then cell lysates were subject to immunoprecipitation with anti-Flag antibodies. The immunoprecipitates were subsequently blotted with indicated antibodies. (D and E) SGC7901 cells were transfected with indicated siRNAs for 48 h. RT-qPCR (D) and Western blot (E) were used to assay the indicated molecules. Mean ± SD of three independent experiments. Two-tailed unpaired t test. (F) SGC7901 cells were transfected with indicated siRNAs for 48 h. Immunofluorescence assays were performed to detect the fluorescent expression of p-ATM (Ser1981) and γH2A.X. Scale bar: 50 μm. ***p < 0.001.

Article Snippet: Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and probed with antibodies against ATM (2873S, Cell Signaling Technology, CST); p-ATM -S1981 (AP0008, ABclonal); METTL3 (15073-1-AP, Proteintech); YTHDF1 (17479-1-AP, Proteintech); YTHDF2 (24744-1-AP, Proteintech); YTHDF3 (25537-1-AP, Proteintech); FTO (TA809392, OriGene); m6A (202003, Synaptic Systems); BRCA1 (20649-1-AP, Proteintech); H2A.X (A11361, ABclonal); γH2A.X (AP0687, ABclonal ); GAPDH(60004-1-Ig, Proteintech); eIF3A (3411T, CST); Flag (F1804, Sigma-Aldrich); anti‐rabbit IgG HRP‐ linked antibody (7074; CST); Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1, Proteintech); α-Tubulin (66031-1-Ig, Proteintech); CHK1(bs-1681R, Bioss); CHK2 (252092, Zenbio); p-CHK2-S19(AP0862, ABclonal); Anti-HA tag monoclonal antibody (TA100012, OriGene); Mouse-IgG (BA1046, Boster); Rabbit IgG (B900610, Proteintech); p21(10355-1-AP, Proteintech); 53BP1 (BA2878, Boster); CDK1 (D160158, BBI life sciences); CDK2 (D199431, BBI life sciences); Cyclin B2 (21644-1-AP, Proteintech).

Techniques: Expressing, Transfection, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Immunofluorescence