Anti gapdh

When organoids reached a density of 70–80% they were treated for 48 h with BaP concentrations that resulted in 30%, 50% and 80% viability. Organoids were then harvested and incubated in TryLE for 10 min at 37 °C to remove the membrane matrix. The organoid pellet was washed with cold PBS and lysed in 62.5 mM Tris (pH 6.8), 1 mM EDTA (pH 8.0), 2% sodium dodecyl sulfate, 10% glycerol, 1X HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA; #78442). Western blotting was carried out as described [48 (link)]. The primary antibodies used were: anti-p21 (1:2000; BD Bioscience, Franklin Lakes, NJ, USA; #BD556431), anti-phospho-H2AX (Ser139, 1:1000; Cell Signalling, Danvers, MA, USA; #9718S), anti-phospho-CHK2 (T68, 1:1000; Cell Signalling, #2197S), anti-phospho-p53 (Ser15, 1:2000; Cell Signalling, #9284S) and anti-GAPDH (1:25,000; Chemicon, Tenecula, CA, USA; #MAB374).

Rabbit antibodies were as follows; anti-BiP from Sigma or a kind gift of Linda M. Hendershot, anti-phospho-eIF2α (Ser51), anti-phospho-PERK and anti-DARPP-32 (a kind gift of Beth Stevens) from Cell Signaling, anti-GFP from Santa Cruz, anti-CHOP and anti-GADD34 were a kind gift of David Ron. Mouse monoclonal antibodies were as follows; anti-GAPDH from Chemicon International, anti-total eIF2α from Cell Signaling and anti-ATF6 from Abnova. Goat anti-mouse IgG conjugated to Cy2 or to dylight 549, goat anti-rabbit IgG conjugated to Cy3 or to dylight 488, and goat anti-rabbit and anti-mouse IgG conjugated to HRP were from Jackson Labs. Normal goat serum was from Vector Laboratories (Burlingame, CA).

Membrane proteins were isolated and Western blot analysis was carried out as described previously [17 (link)]. The protein contents of the samples were determined by the Bradford protein determination method using the Bio-Rad protein assay reagent. The following antibodies were used: Anti-phospho-Akt (Ser473), anti-phospho-ERK1/2, and anti-PKCs (SantaCruz, USA), anti-TG2 (CUB 7402, Biomeda, USA), and anti-GAPDH (Chemicon, USA). Immunoblots were visualized and revealed by autoradiography using the enhanced chemiluminescence ECM detection kit (Amersham Biosciences, UK). Anti-α1 adrenergic receptor antibody [EPR11821(2)] (ab192614) was used, as supplied from Abcam Co. (Cambridge, MA, USA).

Anti JMJD6 (Santa Cruz, sc28349), anti UBF (Bethyl, A301-859A), anti histone H3 (Abcam, Ab1791), anti BrdU (Sigma, B2531), anti gamma H2AX (Cell signaling, 9718(20E3)), anti GAPDH (Chemicon, MAb374), anti myc (Santa Cruz, sc-40), anti Treacle (Santa Cruz, sc374536), anti V5 (Cell Signaling, 12032), anti NBS1 (Sigma, PLA0179), anti phospho ATM (S1981) (Cell signaling, 10H11.E12).Anti Rad51 (Millipore, PC130), Anti RPA S33 (Bethyl, A306-246A-T)

The protein extracts from the cortex and cultured astrocytes were homogenized in 250 mg tissue/0.5 ml cold lysis buffer (see above). Then they were kept on ice for 30 min, centrifuged at maximum speed for 15 min, and the supernatant was collected to determine the proteins levels using the Bradford Assay (Bio-Rad, Hercules, CA, USA). Lysates were separated by SDS-PAGE gels and were transferred to PVDF membranes following standard techniques. Membranes were blocked with 5% non-fat dried milk in TBS containing 0.1% Tween-20 (TBS-T). Next they were then incubated overnight with the following primary antibodies: anti-NLRP3 (1 μg/ml, Abcam), anti-caspase-1 (1/100, Santa Cruz Biotechnology); anti-pro-caspase-1 (1/200, Abcam); anti-Apaf-1 (1 μg/ml, Millipore Bioscience Research Reagents); anti-caspase-3 (1:500) and anti-caspase-9 (1:1000, Cell Signaling).
Some membranes were stripped for 1 h at 60°C in an SDS solution (2% SDS, 0.85% 2-ME, and 65 mM Tris-HCl, pH 6.8, and were washed and incubated with anti-GAPDH (1/3000, Chemicon) for 2 h as a loading control. The intensity of the bands was quantified with the image analysis program, α-Ease FC, version α Imager 2200 (Alpha Innotech Corporation).

Proteins were separated by SDS-PAGE and transferred to Immobilon polyvinylidene difluoride membrane. After blocking of non-specific binding, the membranes were incubated with the first antibodies, followed by incubation with the corresponding secondary antibodies conjugated with alkaline phosphatase, and visualized using BCIP/NBT kit (Nacalai Tesque, Kyoto, Japan). Polyclonal anti-EF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-GAPDH (Chemicon International, Temecula, CA, USA) were used as the first antibodies.

Western blotting was performed as described previously (Hosoi et al., 2015 (link)). Briefly, cells were washed with ice-cold PBS and lysed in buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na₃VO₄, 10 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% NP-40 for 20 min. The lysates were centrifuged at 15,000 rpm for 20 min at 4°C, and supernatants were collected. Samples were boiled with Laemmli buffer for 3 min, fractionated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred at 4°C to nitrocellulose membranes. These membranes were then incubated with anti-phospho STAT3 (Tyr705: Cell Signaling; 1:1,000), anti-STAT3 (Santa Cruz; 1:1,000), anti-phospho STAT5 (Tyr694: Cell Signaling; 1:1,000), and anti-GAPDH (Chemicon; 1:1,000) antibodies, followed by an anti-horseradish peroxidase-linked antibody. Peroxidase binding was detected by chemiluminescence using an enhanced chemiluminescence system (Thermo scientific). Multiple independent experiments were performed and the numbers of the experiments performed was indicated in the figure legends (n = 3∼7).