Anti p eif2α

Cells were grown to 70% confluency in DMEM with 10% FBS, lysed with 1× Passive Lysis Buffer (Promega) and ultrasonicated. Lysates were centrifuged (15,000 × g, 10 min), supernatant harvested and normalized to protein concentration as measured using Micro BCA Protein Assay Kit (Thermo Scientific). The specific antibodies used in this study were: polyclonal anti-myc (Abcam, ab9106); polyclonal anti-RPS2 (Abcam, ab154972); polyclonal anti-RPS9 (Abcam, ab117861); polyclonal anti-tubulin (Abcam, ab6046); polyclonal anti-alpha actin (Abcam, ab5694); polyclonal anti-peIF2α (Cell Signaling, 9721S); anti-eIF2α (Cell Signaling, 9722); polyclonal anti-ubiquitin (Abcam, ab7780); polyclonal anti-GAPDH (Abcam, ab9485); mouse monoclonal anti-p53 (Abcam, ab26); HRP-conjugated goat anti-rabbit (Invitrogen, G-21234); and goat anti-mouse antibodies (Invitrogen, A10551).

Anti-CHOP, anti-caspase-12, anti-caspase-9, anti-caspase-3, anti-caspase-3 cleaved, anti-Bip, anti-p-eIF2α, anti-p-JNK, anti-p-ERK, anti-p-p38, anti-IRE-1α, anti-Cyt C, and anti-COX IV antibodies were purchased from Cell Signaling Technology; anti-α-tubulin antibodies were acquired from Biyuntian (Nanjing, China); anti-Ag85 and anti-GroEL1 were obtained from Abcam (Cambridge, UK); and anti-BAX antibodies were from Santa Cruz Biotechnology. Goat anti-mouse-IgG-HRP (Proteintech, Wuhan, China) and goat anti-rabbit-IgG-HRP (Proteintech) were used as secondary antibodies. Western blots were detected using the Amersham^TM ECL^TM Prime western blotting detection reagent (GE Healthcare, Buckinghamshire, UK) and the ChemiScope 3400 mini imaging system (Clinx Science Instruments, Shanghai, China). The results shown are representative of three independent experiments.

Immunoblotting analysis was performed as previously described [44 ]. The following primary antibodies were used: anti-Claudin-2 (Life Technologies; 325,600), anti-Claudin-3 (Thermo Scientific; PA5-16867), anti-Occludin (Invitrogen; 40-4700), Anti-CREB3L3 (Santa Cruz Biotechnology; SC-69,375), anti-IGFBP5 (Cell Signalling; 10,941S), anti-IGF-1 (Abcam; ab9572), anti-SAPK/JNK (Cell Signalling; 9252T), anti-p-SAPK/JNK (Cell Signalling; 9255S), anti-p-EIF2α (Cell Signalling, 3597S). Secondary antibodies utilised: anti-Mouse (Cell signalling; 7076S), anti-Rabbit (Cell Signalling, 7074S), anti-Goat (Abcam; 7076S). All antibodies were used at a final concentration of 0.1–1 µg/mL. After incubation with the appropriate horseradish peroxidase-conjugated anti-Mouse (GE Healthcare: NA931V) and anti-Rabbit (GE Healthcare: NA934V) IgG secondary antibodies (1:2000 dilution), signals were detected using enhanced chemiluminescence (Pierce, Rockford IL, USA).

Cells were harvested and lysed and protein content of cell lysates was measured as described previously [26 (link)] Whole cell lysates were separated by SDS-PAGE (in 10% or 15% SDS-polyacrilamide gel). SDS-PAGE and Western Blot analysis was done according to our previous study [26 (link)]The following antibodies were applied: anti-NQO1 (Cell Signaling, A180), anti-CyclinD1 (Santa Cruz, A-12), anti-p21 (Santa Cruz, C-19), anti-p27 (Santa Cruz, C-19), anti-p15-INK4b (proteintech, 12877-1-AP), anti-p16-INK4A (proteintech, 10883-1-AP), anti-PERK (Cell Signaling, 3192S), anti-P-eIF2α (Cell Signaling, 9722S9), anti-P-eIF2α (Ser51) (Cell Signaling, 9721L), and anti-GAPDH (Santa Cruz, 6C5) and HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S).

The protein expression was determined using Western blot analysis as described [16 (link)]. Briefly, the kidney tissue and cell lysates were separated on SDS-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membranes. Then, the membranes were incubated with various antibodies including anti-p-eIF2α (1 : 5000, Cell Signaling Technology), anti-p-IRE1α (1 : 4000, Santa Cruz Biotechnology), ATF6α (1 : 5000, Cell Signaling Technology), anti-pJNK (1 : 5000, Cell Signaling Technology), anti-CHOP (1 : 1000, Santa Cruz Biotechnology), anti-pSrc (1 : 1000; Cell Signaling Technology), anti-pFyn (1 : 1000; Santa Cruz Biotechnology), and anti-β-actin (1 : 1000) overnight at 4°C on a shaker. Then, the membranes were incubated with respective secondary antibodies, washed, and reacted with an enhanced chemiluminescent sensitive plus reaction (BioFX Laboratories, Inc., Owings Mills, MD, USA). The bands were quantified by using ImageJ software and normalized by β-actin.

The total protein of PDLCs and P-PDLCs or mouse gingiva was extracted, and 20 μg protein of each sample was separated and blotted onto a polyvinylidene fluoride membrane (Millipore, USA). Then, membranes were blocked and incubated with primary antibodies including anti-actin (Abcam, UK; ab3280, 1 : 10000), anti-cleaved Caspase-3 (Cell Signaling Technology, USA; #9664, 1 : 1000), anti- PPARγ2 (Abcam, UK; ab45036, 1 : 1000), anti-HSP70 (Zen, China; 200304, 1 : 1000), anti-TRPA1 (Novus, USA; NB100-91319, 1 : 1000), anti-SOD1 (Abcam, UK; ab51254, 1 : 1000), anti-SOD2 (Santa Cruz, USA; sc-133254, 1 : 1000), anti-PERK (Cell Signaling Technology, USA; #5683, 1 : 1000), anti-CHOP (Cell Signaling Technology, USA; #2895, 1 : 1000), anti-ATF-4 (Cell Signaling Technology, USA; #11815, 1 : 1000), anti-eIF2α (Cell Signaling Technology, USA; #5324, 1 : 1000), and anti-p-eIF2α (Cell Signaling Technology, USA; #3398, 1 : 1000) at 4°C overnight. The membranes were washed and incubated with secondary antibody (1 : 10,000) and were visualized by the Image Quant Tanon-5200Multi (Tanon, China). The relative intensity of the protein was quantitatively analyzed by the ratio of actin, and all assays were repeated three times.

Cells were grown on coverslips overnight and treated with 0.5 mM H₂O₂ (Sigma-Aldrich) for 3 h or 250 μM cisplatin (Beyotime) for 4 h to induce SGs. After rinsing with PBS, cells were fixed in 4% paraformaldehyde for 8 min at RT, rinsed by PBS for three times, followed by incubation in 0.2% Triton X-100 for 8 min at RT and rinsed for three times with PBS. The coverslips were then blocked by 2% BSA for 1 h at RT and incubated with primary antibodies at 4 °C overnight. Primary antibodies used: anti-G3BP1 (1:200; Proteintech), anti-p-eIF2α (1:100; Cell Signaling Technology), anti-YBX1 (1:100; Cell Signaling Technology), anti-APE1 (1:100; Santa Cruz Biotechnology) and anti-Flag-tag (1:100; GeneScript). After rinsing with PBS, coverslips were incubated with Alexa Fluor 488-conjuaged secondary antibody (1:100; Thermo Fisher) or Alexa Fluor 674-conjugated secondary antibody (1:100; YiSheng) at RT for 1 h. Coverslips were then rinsed with PBS for three times and rinsed with ddH₂O, dried and mounted in Prolong Glass Antifade Mountant (with Hoechst 33342; Invitrogen). Images were captured using a Nikon fluorescent microscope and analyzed by Image J software. Cells containing more than 2 SG foci were counted as SG-containing cells [42 (link)].